WO2009091053A1 - Crystal production method, frozen crystal production method, crystal, crystal structure analysis method, crystallization screening method, and crystallization screening apparatus - Google Patents

Crystal production method, frozen crystal production method, crystal, crystal structure analysis method, crystallization screening method, and crystallization screening apparatus Download PDF

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Publication number
WO2009091053A1
WO2009091053A1 PCT/JP2009/050592 JP2009050592W WO2009091053A1 WO 2009091053 A1 WO2009091053 A1 WO 2009091053A1 JP 2009050592 W JP2009050592 W JP 2009050592W WO 2009091053 A1 WO2009091053 A1 WO 2009091053A1
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Prior art keywords
crystal
gel
crystallization
biological material
solution
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PCT/JP2009/050592
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French (fr)
Japanese (ja)
Inventor
Shigeru Sugiyama
Hiroaki Adachi
Hiroyoshi Matsumura
Kazufumi Takano
Satoshi Murakami
Tsuyoshi Inoue
Yusuke Mori
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Sosho, Inc.
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Priority to JP2009550072A priority Critical patent/JP5351771B2/en
Publication of WO2009091053A1 publication Critical patent/WO2009091053A1/en

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    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B5/00—Single-crystal growth from gels

Definitions

  • the present invention relates to a crystal production method, a frozen crystal production method, a crystal, a crystal structure analysis method, a crystallization screening method, and a crystallization screening apparatus.
  • a method for producing a crystal that precipitates (crystallizes) a crystal from a solution of protein, nucleic acid or the like requires a very advanced technique and is difficult. Furthermore, crystals of proteins, nucleic acids, and the like are very brittle and are likely to be distorted inside the crystals, so that there is a problem that they are easily destroyed unless handled carefully.
  • an object of the present invention is to provide a method for producing a crystal, in which a crystal can be easily produced and the produced crystal is easy to handle.
  • the inventors of the present invention have made extensive studies to solve the above problems. As a result, it has been found that in the method for producing crystals of biological materials such as proteins and nucleic acids, crystals are precipitated from the gel instead of the biological material solution. As described above, in the method for producing crystals of biological substances such as proteins and nucleic acids, conventionally, crystals are generally precipitated from a solution. The inventors of the present invention have made the first attempt to deposit crystals directly from the gel in these biological substances.
  • the crystal production method of the present invention is: A method for producing a crystal of biological material, The method includes a crystallization step of crystallizing the biological material in a gel.
  • the present invention provides: A method for producing a frozen crystal comprising a freezing step of freezing a crystal of biological material,
  • the method for producing a crystal according to the present invention further includes a coated crystal production process for producing the coated crystal coated with the gel, In the freezing step, a frozen crystal production method is provided, wherein the coated crystal is frozen.
  • the present invention provides: A method for producing a crystal of a biological material, comprising a crystal growth step of further growing a biological material crystal that is a seed crystal in a solution of the biological material, In the crystal growth step, there is provided a crystal manufacturing method, wherein the seed crystal is a coated crystal coated with the gel manufactured by the crystal manufacturing method of the present invention.
  • this crystal production method may be referred to as “growth crystal production method of the present invention” or simply “growth crystal production method”.
  • the present invention provides a crystal produced by the crystal production method of the present invention, the frozen crystal production method of the present invention, or the growth crystal production method of the present invention.
  • the present invention provides: A method for structural analysis of a crystal of a biological material, There is provided a structural analysis method comprising a structural analysis step of structural analysis of the coated crystal manufactured by the crystal manufacturing method of the present invention or the frozen crystal manufactured by the frozen crystal manufacturing method of the present invention.
  • the present invention provides: A crystallization screening method for screening crystallization conditions of a biological material, A crystal production process for producing crystals of the biological material; Including a screening step of screening crystallization conditions in the crystal manufacturing step, In the crystal production step, a crystallization screening method is provided, wherein the crystal is produced by the crystal production method of the present invention or the frozen crystal production method of the present invention.
  • the present invention provides: A crystallization screening apparatus for screening crystallization conditions of a biological material, Crystal manufacturing means for manufacturing crystals of the biological material; Screening means for screening the crystallization conditions of the biological material,
  • the crystallization screening apparatus is characterized in that the crystal production means includes a crystallization means for precipitating crystals of the biological material in a gel.
  • the crystal manufacturing method of the present invention it is possible to easily manufacture a crystal of the biological substance, rather than precipitating the crystal from a solution.
  • the generated crystal is fixed by a gel. More specifically, for example, since the generated crystal is fixed to the gel, it is possible to prevent damage due to collision with the wall of the container, polycrystallization due to collision between the generated crystals, and the like. It is thought that it is easy to grow into a few single crystals.
  • these considerations are merely examples of possible mechanisms and do not limit the present invention in any way.
  • the crystal is not easily damaged by freezing. Furthermore, according to the structure analysis method of the present invention, since the structure analysis of the coated crystal coated with the gel or the frozen crystal frozen from the gel is performed, there is an advantage that the crystal is easy to handle.
  • the crystal of the present invention is manufactured by the crystal manufacturing method of the present invention, the frozen crystal manufacturing method of the present invention, or the grown crystal manufacturing method of the present invention, so that it is suitable for crystal structure analysis, for example. It has special characteristics.
  • the manufacturing method of the crystal of the present invention is not particularly limited, and may be a crystal manufactured by any other manufacturing method as long as it has similar characteristics.
  • the crystallization screening method of the present invention can easily produce crystals by the crystal production method of the present invention, it is not necessary to set the crystal production conditions so strictly, and thus screening can be performed easily. . Furthermore, the crystallization screening apparatus of the present invention can simplify the configuration of the apparatus for the same reason.
  • FIG. 1A is a photograph of a crystal obtained according to an embodiment of the present invention.
  • FIG. 1B is a photograph of the crystal of FIG. 1A taken at a different angle.
  • FIG. 2A is a diagram showing an X-ray diffraction image of the crystal of FIG.
  • FIG. 2B is an enlarged view of a part of FIG. 2A.
  • FIG. 3A is a diagram showing an X-ray diffraction image of a crystal obtained by another example of the present invention.
  • FIG. 3B is an enlarged view of a part of FIG. 3A.
  • FIG. 4 is a diagram showing an X-ray diffraction image when the crystal of FIG. 3 is measured under different conditions.
  • FIG. 1A is a photograph of a crystal obtained according to an embodiment of the present invention.
  • FIG. 1B is a photograph of the crystal of FIG. 1A taken at a different angle.
  • FIG. 2A is a diagram showing an X-ray diffraction image of
  • FIG. 5A is a diagram showing an X-ray diffraction image when the crystal of FIG. 4 is measured under still another condition.
  • FIG. 5B is an enlarged view of a part of FIG. 5A.
  • FIG. 6A is a photograph showing a state immediately before the crystals of FIGS. 3 to 5 are immersed in the cryoprotectant.
  • FIG. 6B is a photograph showing a state after 5 seconds from immersing the crystal of FIG. 6A in the cryoprotectant.
  • FIG. 6C is a photograph showing a state one minute after immersing the crystal of FIG. 6A in the cryoprotectant.
  • FIG. 6D is a photograph showing a state after 5 minutes from immersing the crystal of FIG. 6A in the cryoprotectant.
  • FIG. 6A is a diagram showing an X-ray diffraction image when the crystal of FIG. 4 is measured under still another condition.
  • FIG. 5B is an enlarged view of a part of FIG. 5A.
  • FIG. 7A is a photograph of a crystal obtained by yet another example of the present invention.
  • FIG. 7B is a diagram showing an X-ray diffraction image of the crystal of FIG. 7A.
  • FIG. 7C is an enlarged view of a part of FIG. 7B.
  • FIG. 8A is a photograph of a crystal obtained according to yet another example of the present invention.
  • FIG. 8B is a diagram showing an X-ray diffraction image of the crystal of FIG. 8A.
  • FIG. 8C is an enlarged view of a part of FIG. 8B.
  • FIG. 9A is a diagram showing a state immediately before the crystal of the comparative example is immersed in the cryoprotectant.
  • FIG. 9B is a diagram showing a state immediately after the crystal of FIG.
  • FIG. 10A is a diagram showing a state immediately before dipping a crystal of another comparative example in an antifreezing agent.
  • FIG. 10B is a diagram showing a state immediately after the crystal of FIG. 10A is immersed in an anti-freezing agent.
  • FIG. 11 is a diagram showing photographs and data of crystals of still another example and comparative example of the present invention.
  • FIG. 12 is a diagram showing an X-ray diffraction image of a crystal according to still another example of the present invention.
  • FIG. 13 is a schematic diagram illustrating an example of a result of crystal structure analysis of a biomolecule.
  • FIG. 14 is a schematic diagram showing still another example of the result of analyzing the crystal structure of a biomolecule.
  • FIG. 15 is a graph showing the crystallization screening result in one example of the present invention.
  • FIG. 16 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 17 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 18 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 19 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 20 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 21 is a graph showing a crystallization screening result in still another example of the present invention.
  • FIG. 22 is a photograph and a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 23 is a photograph and a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 24 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 25 is a photograph showing a crystallization screening result in still another example of the present invention.
  • FIG. 26 is a photograph showing an E. coli-derived protein crystal in still another example of the present invention.
  • FIG. 27 is a photograph showing a DNA crystal in still another example of the present invention.
  • FIG. 28 is a graph showing a crystallization screening result and a photograph of a crystal in still another example of the present invention.
  • FIG. 29 is a photograph showing an example in which the coated gel is removed by lowering the temperature.
  • FIG. 30 is a photograph showing the drying tolerance evaluation of the crystals of the example and the crystals of the comparative example.
  • FIG. 31A is a photograph showing the drying tolerance evaluation of crystals of still another example and comparative example.
  • FIG. 31B is a photograph showing the drying tolerance evaluation of the crystals of the same example and the comparative example after the elapse of the evaluation time of FIG. 31A.
  • FIG. 32 is a photograph showing crystals before and after gelation of the surrounding solution in crystals produced in yet another example.
  • FIG. 33 is a diagram showing an X-ray diffraction image of the crystal of the example.
  • FIG. 34 is a diagram showing an X-ray diffraction image of a crystal of still another example.
  • FIG. 35 is a photograph showing processing of a gel-coated crystal with a femtosecond laser.
  • FIG. 36 is a photograph showing removal of a gel-coated crystal that has been subjected to femtosecond laser processing.
  • FIG. 37 shows a photograph of the crystal of FIG. 36 and X-ray crystal structure analysis data.
  • FIG. 38 is a photograph showing one example of a method for producing a grown crystal.
  • FIG. 39 is a photograph showing another example of the method for producing a grown crystal.
  • FIG. 40 is a diagram schematically illustrating an example of a crystallization screening method using a concentration gradient by centrifugation.
  • FIG. 41 is a photograph showing a crystallization screening result by the method of FIG. FIG.
  • FIG. 42 is a schematic view illustrating the crystal mounting operation in the crystal structure analysis.
  • FIG. 43 is a schematic view illustrating an apparatus used for a crystal mounting operation in crystal structure analysis.
  • FIG. 44 is a schematic diagram showing an example of the crystallization screening apparatus of the present invention.
  • FIG. 45 is a schematic diagram showing still another example of the crystallization screening apparatus (crystallization kit) of the present invention.
  • FIG. 46 is a schematic diagram showing an example of crystallization screening according to the present invention.
  • FIG. 47 is a graph showing an example of a gel strength measurement result of an agarose gel.
  • FIG. 48 is a graph obtained by extracting a part of the data of the graph of FIG.
  • the crystal production method of the present invention is a method for producing a crystal of a biological material, which includes a crystallization step of crystallizing the biological material, wherein the biological material is crystallized in a gel in the crystallization step. It is characterized by making it.
  • the “biological substance” may be a biological substance, but may be a synthetic substance having the same structure as it, or a derivative or an artificial substance having a structure similar to the biological substance.
  • the biological substance when it is a biopolymer compound, it may be a polymer compound derived from a living body, a synthetic polymer compound having the same structure as it, or a derivative or artificial material having a structure similar to that of a bio-derived polymer compound. It may be a polymer compound.
  • the biological material is a protein, it may be a biologically derived (naturally-derived) protein, a synthetic protein, a naturally occurring natural protein, or a non-naturally occurring artificial protein.
  • the biological substance when it is a peptide, it may be a biologically derived (naturally-derived) peptide, a synthetic peptide, a naturally occurring peptide having a naturally occurring structure, or an artificial peptide having a non-naturally occurring structure.
  • the biological material when it is a nucleic acid, it may be a biologically derived (naturally-derived) nucleic acid, a synthetic nucleic acid, a naturally-occurring natural nucleic acid, or a non-naturally-occurring artificial nucleic acid.
  • the biological substance when it is a sugar chain, it may be a sugar chain derived from a living body (naturally derived), a synthetic sugar chain, a natural sugar chain having a naturally occurring structure, or an artificial sugar chain having a non-naturally occurring structure.
  • the biological material is not particularly limited, but for example, a biopolymer compound, protein, natural protein, artificial protein, peptide, natural peptide, artificial peptide, nucleic acid, natural nucleic acid, artificial nucleic acid, sugar chain, natural sugar chain, or artificial A sugar chain is preferred.
  • the natural nucleic acid is not particularly limited, and examples thereof include DNA and RNA.
  • the artificial nucleic acid is not particularly limited, and examples thereof include LNA and PNA.
  • the molecular weight of the biological material is not particularly limited.
  • the molecular weight of the “biopolymer compound” is, for example, 5000 or more, but is not limited thereto, and may be less than 5000.
  • the molecular weight is, for example, 1000 or more, but is not limited thereto, and may be less than 1000.
  • the crystal production method of the present invention further includes a solution preparation step of preparing a solution of the biological material prior to the crystallization step, and a gelation step of preparing the gel by gelling the solution.
  • the biological material solution further contains a gelling agent.
  • the gelling agent is not particularly limited, but is preferably at least one selected from the group consisting of polysaccharides, thickening polysaccharides, proteins, and gels at elevated temperature, agarose, agar, More preferably, it is at least one selected from the group consisting of carrageenan, gelatin, collagen, polyacrylamide, and gelled polyacrylamide gel at elevated temperature.
  • the gelation temperature is not particularly limited, but is, for example, 0 to 90 ° C., preferably 0 to 60 ° C., more preferably 0 to 35 ° C. from the viewpoint of ease of crystal production.
  • the gelling agent may be, for example, a gel that gels at a low temperature and forms a sol at a high temperature, or a gel that forms a sol at a low temperature and gels at a high temperature. Gels that sol at a low temperature and gel at a high temperature are referred to as “gels at elevated temperature”. Further, for example, a gel that returns to a sol again when the temperature of the gel obtained by cooling is increased again or when the temperature of the gel obtained by increasing temperature is cooled again is preferable. Such a gelling agent is called “thermo-reversible gel”.
  • the gelling agent may be, for example, a hydrogel or an organogel, but is preferably a hydrogel.
  • the hydrogel for example, it is more preferable to use a gelled hydrogel at elevated temperature.
  • the gelled hydrogel at elevated temperature has the property of solling at a low temperature and gelling at a high temperature as described above, contrary to a general gel that gels at a low temperature and sols at a high temperature.
  • the coated crystal coated with the gelled hydrogel at elevated temperature has advantages such as being particularly strong in drying and capable of easily removing the gel by cooling.
  • limit especially as a gelatinization type hydrogel at the time of temperature rising For example, a meviol gel is mentioned.
  • Mebiol gel is a trade name of a gelled hydrogel produced by Mebiol Co., Ltd. and has the following chemical structure, for example.
  • Meviol gel is a polyacrylamide gel having properties as a gelling hydrogel at elevated temperature and properties as a thermoreversible hydrogel.
  • crystal production method of the present invention can be performed, for example, as follows.
  • Solution preparation step First, the biological material is dissolved in a solvent to obtain a solution.
  • the said solvent is not restrict
  • Specific examples of the solvent include water, ethanol, methanol, acetonitrile, acetone, anisole, isopropanol, ethyl acetate, butyl acetate, chloroform, cyclohexane, diethylamine, dimethylacetamide, dimethylformamide, toluene, butanol, and butyl methyl ether.
  • the concentration of the biological substance is not particularly limited, but is, for example, 0.2 to 300 mg / mL, preferably 0.5 to 100 mg / mL, more preferably 1 to 50 mg / mL.
  • a gelling agent is added to the biological material solution to cause gelation, thereby preparing a gel containing the biological material.
  • the gelling agent may be added directly to the biological material solution, but it is preferable to prepare a gelling agent solution separately and then mix it with the biological material solution because it is easy to mix uniformly.
  • the solvent of the gelling agent solution is not particularly limited, but is the same as the biological material solution, for example.
  • the concentration of the gelling agent in the gelling agent solution is not particularly limited, but from the viewpoint of gel strength and the like described later, for example, 0.6 to 50% by mass, preferably The amount is 0.8 to 40% by mass, more preferably 1.0 to 30% by mass, still more preferably 1.0 to 25% by mass, and particularly preferably 1.0 to 20% by mass.
  • the mechanism of the correlation between the gelling agent concentration and the ease of crystallization of the biological substance is unknown, but for example, the gelling agent concentration can be obtained by utilizing the crystallization screening method of the present invention described later. Can be set as appropriate.
  • the method of gelling after adding a gelling agent to the biological material solution is not particularly limited.
  • the gelling agent solution may be prepared at a temperature higher than the gelation temperature (for example, 20 to 45 ° C.), mixed with the biological material solution, and then allowed to stand at a temperature equal to or lower than the gelation temperature. More specifically, for example, after the gelling agent solution and the biological material solution are mixed, they may be sealed in a capillary and gelled in the capillary. As a result, the gel is sealed in the capillary.
  • the gelling agent solution may be prepared at a low temperature, mixed with the biological material solution, and then gelled by increasing the temperature.
  • the gel strength after the gelation is, for example, 100 Pa or more, preferably 200 Pa or more, more preferably 300 Pa or more, from the viewpoint of easy protection of the biological material crystals deposited in the crystallization step described below from physical impact. Preferably it is 500 Pa or more, Most preferably, it is 1000 Pa or more.
  • the gel strength of the gelling agent is preferably as high as possible, and the upper limit is not particularly limited, but is, for example, 200,000 Pa or less.
  • the gel strength after gelation can be appropriately set by adjusting the gelling agent concentration.
  • the gel strength at the same gelling agent concentration varies depending on the type of the gelling agent. For example, as shown in the graph of FIG.
  • agarose III agarose SP (agarose sea plaque), and agarose 9A (all trade names of Takara Bio Inc.) are usually agarose after gelation at the same concentration.
  • the gel strength increases in the order of III> agarose SP> agarose 9A.
  • the graph of FIG. 48 shows the measurement results of only agarose 9A in FIG. 47 and 48, the horizontal axis represents the concentration (mass%) of each agarose, and the vertical axis represents the gel strength (g / cm 2 ). Note that 1 g / cm 2 corresponds to 98.0665 Pa. Further, when the gelling agent concentration is less than a certain critical concentration, the gel strength cannot be measured (0 on the graph) because the solution containing the gelling agent does not clearly gel.
  • the concentration of the gelling agent is not less than the critical concentration.
  • the critical concentration is about 0.6% by mass as shown in FIG. 47 and FIG.
  • the gel strength is a numerical value measured at a frequency of 1 Hz and a measurement temperature of 20 ° C. using a rheostress RS1 Rheometer (trade name of Rheometer manufactured by Eihiro Seiki Co., Ltd.) in a dynamic viscoelasticity measurement mode.
  • the measured values shown in FIGS. 47 and 48 were also measured by this method.
  • the gel strength is lower than the gel dissolution temperature and can be divided by 5 (example: : 15 ° C, 10 ° C, 5 ° C, 0 ° C, -5 ° C).
  • the gel strength is higher than the gel dissolution temperature and is divisible by 5 degrees Celsius.
  • the measured value at the lowest measurable temperature among the temperatures (example: 25 ° C, 30 ° C, 35 ° C) is used.
  • this measurement method is an example of the measurement method of the gel strength, and the present invention is not limited by the steps and conditions in this measurement method. Further, even if other rheometers are used, the same gel strength measurement value can be obtained except for errors if measurement is performed in the same measurement mode, measurement temperature and frequency.
  • Crystallization step Further, crystals of the biological material are precipitated from the gel.
  • This method is not particularly limited.
  • the gel may be left still and wait for crystals of the biological material to precipitate.
  • the gel may be allowed to stand in contact with or in close proximity to the precipitant solution. More specifically, for example, after the gel is sealed in a capillary, it may be immersed in the precipitant solution.
  • the precipitating agent is not particularly limited, and for example, the same precipitating agent as used in a known crystal production method may be used.
  • the precipitating agent examples include sodium chloride, calcium chloride, sodium acetate, ammonium acetate, ammonium phosphate, ammonium sulfate, potassium sodium tartrate, sodium citrate, PEG (polyethylene glycol), magnesium chloride, sodium cacodylate, HEPES (2 -[4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), MPD (2-methyl-2,4-pentanediol), Tris-HCl (trishydroxymethylaminomethane hydrochloride) Is at least one.
  • the solvent for the precipitating agent solution is not particularly limited, and may be the same as the biological material solution, for example.
  • the concentration of the precipitating agent is not particularly limited, but is, for example, 0.0001 to 10M, preferably 0.0005 to 8M, and more preferably 0.0005 to 6M.
  • the said precipitating agent may contain the pH adjuster etc. suitably as needed.
  • the precipitating agent may also be referred to as “precipitating agent”.
  • the method of bringing the gel containing the biological material solution into contact with or in proximity to the precipitant solution is described.
  • the crystallization method using the precipitant is not limited thereto.
  • the biological material solution is mixed with a precipitating agent together with a gelling agent, gelled to form a gel containing the precipitating agent, and left as it is after gelation to precipitate crystals.
  • the method may be used. Such a crystallization method is called “batch method”.
  • the solvent of the biological material solution, the concentration of the biological material, the concentration of the gelling agent, the concentration of the precipitating agent and the like are not particularly limited, but are the same as described above, for example.
  • Crystallization of biological materials such as proteins can be controlled by, for example, the mixing ratio of the biological material and the precipitating agent.
  • the mixing ratio in more detail, for example, it is possible to obtain a higher quality and larger single crystal.
  • a concentration gradient can be formed by diffusing a precipitating agent as described above in a gel having a network molecular structure (for example, agarose gel).
  • the diffusion rate of the precipitating agent is sufficiently slower than the crystal growth rate.
  • the production method of the present invention can also be applied to a crystallization screening method as a “combinatorial crystallization technique” capable of searching a mixture ratio of various combinations all at once.
  • a specific screening method using the concentration gradient formation and an apparatus used therefor are as described below, for example.
  • the crystal manufacturing method of the present invention can be carried out as described above, the present invention is not limited to this.
  • the crystal production method of the present invention may be performed, for example, by the following method (i) or (ii), or by other methods.
  • the following (i) is referred to as “counter diffusion method”, and the following (ii) is referred to as “liquid-solid method”.
  • the gelling agent solution is prepared at a temperature higher than the gelation temperature (for example, 20 to 45 ° C.), mixed with the solution of the biological material (protein etc.), and enclosed in a capillary. To gel. Thereafter, it is brought into contact with a precipitant solution as necessary.
  • a gel for example, solid agarose
  • a solution of the biological material protein, etc.
  • the precipitating agent may be used in the form of a solution or as a solid.
  • the biological material is crystallized in a gel.
  • This “crystallize in gel” may crystallize the biological material in the gel after the gelation step, for example, as described above.
  • the term “crystallize in gel” means, for example, that the biological material solution containing a gelling agent is not gelled and is in a sol state, the biological material in the gelling agent solution in the sol state. And crystallizing.
  • the mechanism is not clear, even if the solution is in a sol state, the inclusion of the gelling agent may lead to the effect of the present invention in that crystals are likely to precipitate and damage to the crystals is less likely to occur. .
  • the method for crystallizing the biological material in the gelling agent solution in the sol state is particularly useful when, for example, the gelling agent is a thermoreversible hydrogel.
  • the gelling agent is a thermoreversible hydrogel.
  • the biological material is crystallized, and then the solution is gelled, and the biological material crystal is further grown in the gel. It is particularly preferred that By stirring the sol-state solution, for example, the biological material crystals are more likely to grow and large crystals can be easily obtained.
  • the stirring speed is not particularly limited, but is, for example, 10 to 1000 rpm, preferably 30 to 200 rpm, more preferably 50 to 100 rpm.
  • the stirring time is not particularly limited, but is, for example, 5 minutes to 60 days, preferably 30 minutes to 30 days, and more preferably 1 hour to 20 days.
  • the temperature of the sol solution during the stirring is not particularly limited, but is, for example, 0 to 40 ° C., preferably 2 to 30 ° C., and particularly preferably 4 to 25 ° C.
  • the crystal produced by the crystal production method of the present invention is preferably, for example, a coated crystal coated with the gel.
  • the production conditions of the coated crystal are not particularly limited.
  • the crystal of the biological material is precipitated from the gel, so that the coated crystal covered with the gel is necessarily formed. Also good.
  • crystals of biological materials such as proteins are brittle and are easily altered by drying or the like. For this reason, biological material crystals are damaged by physical impact or drying unless they are operated carefully and quickly, for example, when they are used as samples for crystal structure analysis (mounting) and when they are used as seed crystals (seeding). In many cases, it will cause alteration.
  • the coated crystal in the present invention is improved in resistance to drying and physical impact because the crystal is coated with a gel, so that the crystal is hardly deteriorated or damaged. For this reason, for example, the mounting operation and the seeding operation are extremely easy to perform. Furthermore, the preservability of crystals is improved.
  • the gel covering the crystal is preferably removed in advance if there is a problem such as causing measurement noise in the crystal structure analysis described later.
  • the removal method is not particularly limited.
  • the gel is a thermoreversible hydrogel, as described above, the gel is made into a sol by cooling and can be easily removed.
  • the cooling temperature for making the sol is not particularly limited, but in the case of meviol gel, for example, it is 15 ° C. or lower.
  • the coated crystal can be processed by an appropriate method, and only the crystal not containing the gel can be taken out.
  • the processing method is not particularly limited, for example, there is processing using laser light.
  • the laser light is not particularly limited, but it is particularly preferable to use a femtosecond laser.
  • the femtosecond laser can be processed only in the vicinity of the condensing point, and thus has an advantage that it can be easily processed while the crystal manufacturing (growing) container is sealed, for example.
  • a crystal can be cut into a size and shape suitable for applications such as structural analysis.
  • the crystal having the appropriate size and shape can be manufactured by cutting the crystal into an appropriate size and shape by such a processing step.
  • the processing step it is possible to appropriately remove only the gel from the coated crystal and manufacture a processed crystal that leaves only the crystal portion. That is, the processed crystal may be a coated crystal coated with a gel or may not be coated with a gel.
  • a high-quality and large-sized crystal can be obtained with a higher probability than in the past, and according to the screening method (concentration gradient method) using the above-described concentration gradient formation, A wide range of search conditions (combinatorial search) is possible. With respect to these, for example, it is possible to obtain results superior to the vapor diffusion method that has been conventionally most commonly used for crystallization of proteins and the like.
  • the following advantages can be obtained by precipitating crystals of the biological material from the gel.
  • the crystal obtained in the solution freely moves in the solution when the crystal is mounted, it may be damaged before the X-ray measurement by indirect or direct contact with the loop or the like. For this reason, in order to perform measurement with high accuracy, a skilled skill in handling the loop is required.
  • the crystal production method of the present invention crystals of the biological substance (proteins, etc.) are precipitated from the gel.
  • the said biological material crystal will be in the state fixed with the gel. Therefore, the mounting operation is easy and the mounting operation is highly reproducible due to the fact that the biological material crystal is fixed by the gel and the movement is inhibited, and the biological material crystal is protected by the gel and is not easily damaged. According to this, for example, by automating the crystal mounting process, it is possible to achieve full automation of X-ray structural analysis of protein crystals and the like, which could not be achieved conventionally.
  • the obtained crystals are covered with the gel, so that the crystals can be frozen and a simple mounting operation can be performed. High accuracy data can be obtained because it can be frozen without degrading quality.
  • the frozen crystal manufacturing method of the present invention is a frozen crystal manufacturing method including a freezing step of freezing the crystal of biological material, as described above, and is coated with the gel by the crystal manufacturing method of the present invention.
  • the method further comprises a coated crystal manufacturing step for manufacturing the coated crystal, wherein the coated crystal is frozen in the freezing step.
  • the method for producing frozen crystals of the present invention is not particularly limited, but it is preferable that the method further includes an immersion step of immersing the coated crystals in an antifreezing agent prior to the freezing step.
  • a frozen state for example, 100K or less.
  • the damage to the crystal due to radiation for example, synchrotron radiation for obtaining high resolution data
  • the freezing instead of the encapsulation in the capillary suppresses damage to the crystal due to radiation.
  • the coated crystal and the frozen crystal are protected with a gel.
  • the gel is considered to suppress damage caused by X-rays or the like.
  • crystallization damage by freezing is further suppressed by further including the immersion process which immerses the said covering crystal
  • the cryoprotectant is not particularly limited, but is preferably an organic compound, such as glycerol (glycerin), MPD (2-Methyl-2,4-PentaneDiol), DMSO (dimethyl sulfoxide), It is at least one selected from the group consisting of PEG (polyethylene glycol) and lithium acetate. These may be used as they are or as an aqueous solution.
  • the amount (concentration) of the cryoprotectant added to the crystallization mother liquor is finely adjusted, and optimal conditions are screened. It was necessary.
  • the concentration of the cryoprotectant is not particularly limited. As described above, according to the present invention, the crystal damage caused by the cryoprotectant can be greatly suppressed. For example, 100% DMSO, PEG, or a high concentration aqueous solution thereof (for example, 50 to 60% by mass) can be used. Crystals of the biological material can also be directly immersed.
  • the type of anti-freezing agent itself may be, for example, the same as that conventionally used.
  • the cryoprotectant molecule may bind to the active site of the biological material and remain in the crystal.
  • This figure is a schematic diagram showing an example of a structural analysis result when glycerol is used as an anti-freezing agent and measurement is performed at a resolution of 0.94 mm in X-ray crystal structure analysis of glucose isomerase (GI).
  • GI glucose isomerase
  • 2GLK in the figure is a PDB ID (protein data bank registration number), which means glucose isomerase (GI).
  • the coated crystal of the present invention when used, for example, as shown in the schematic diagram of FIG. 14, the freezing structure is not bonded to the active site and free structure information is obtained, which is advantageous for drug design and the like. .
  • the crystal damage due to the cryoprotectant is significant, and therefore the types of antifreeze agents that are less likely to cause crystal damage have been limited. For this reason, it has been difficult to select an anti-freezing agent that hardly remains in the crystal from among the anti-freezing agents that hardly cause crystal damage.
  • the damage of the crystal caused by the cryoprotectant is suppressed, so that the range of the cryoprotectant that can be selected is wide, and the cryoprotectant that does not easily remain in the crystal is appropriately selected. You can also do it.
  • glucose isomerase although not necessarily clear, it is considered that lithium acetate is less likely to remain in the crystal than glycerol.
  • this explanation is merely an example, and does not limit the present invention at all. For example, it does not mean that glycerol is inappropriate as an antifreezing agent used in the present invention.
  • the growth crystal manufacturing method of the present invention includes a crystal growth step of further growing a biological material crystal as a seed crystal in a solution of the biological material, and the seed crystal is coated with the gel. It is.
  • the growth crystal production method of the present invention is not particularly limited.
  • various conditions such as the concentration and temperature of the biological material solution in the crystal growth step may be appropriately set with reference to a method of growing a crystal using a seed crystal in the prior art.
  • the preferable concentration of the biological material solution varies depending on the type of the biological material, but is preferably as high as possible from the viewpoint of ease of crystallization of growth.
  • the temperature is not particularly limited, and may be left at room temperature (about 5 to 35 ° C.), or may be cooled to a low temperature of 5 ° C. or lower or 0 ° C. or lower with a refrigerator or the like.
  • the time for the crystal growth step is not particularly limited, and may be allowed to stand as appropriate until a desired crystal is precipitated.
  • a crystal manufacturing method for further growing a seed crystal in a solution is widely performed.
  • a biological material such as a protein
  • the crystal since the crystal is brittle, a physical impact is caused when the seed crystal is moved into the biological material solution. There was a risk of damage.
  • the seed crystal after the seed crystal is moved into the biological material solution, the seed crystal may be dissolved due to a change in the concentration or osmotic pressure of the solution around the seed crystal, and crystal growth may not occur. It was.
  • the growth crystal manufacturing method of the present invention since the seed crystal is a coated crystal coated with the gel, the risk of damage due to physical impact is drastically reduced.
  • the seed crystal is a coated crystal coated with the gel, dissolution of the seed crystal is also suppressed, and the crystal is likely to grow.
  • a large crystal suitable for various applications such as neutron beam crystal structure analysis can be obtained with high reliability by a simple operation.
  • the structural analysis method of the present invention is a method for structural analysis of a crystal of a biological material, which is manufactured by a coated crystal manufactured by the crystal manufacturing method of the present invention or a frozen crystal manufacturing method of the present invention. And a structural analysis step for structural analysis of the frozen crystals.
  • a method for analyzing the structure of the crystal is not particularly limited, but it is preferable to use X-ray crystal structure analysis or neutron beam crystal structure analysis. According to the crystal manufacturing method of the present invention, as described above, damage to the crystal, crystallization, and the like can be prevented, and thus a large crystal suitable for neutron beam crystal structure analysis can be obtained.
  • the structure analysis method of the present invention is not particularly limited, and may be performed in the same manner as the conventional crystal structure analysis method, X-ray crystal structure analysis method, or neutron beam crystal structure analysis method.
  • Crystals of biological substances (proteins etc.) subjected to structural analysis in the structural analysis method of the present invention are coated crystals manufactured by the crystal manufacturing method of the present invention or frozen crystals manufactured by the frozen crystal manufacturing method of the present invention. High quality and large crystals.
  • highly accurate data for example, X-ray diffraction data
  • the frozen crystal production method of the present invention there are advantages as described in the section of the crystal production method and the frozen crystal production method of the present invention.
  • the structure analysis method of the present invention is not particularly limited as described above, and any method may be used.
  • the structure analysis method can be performed as follows.
  • the first scheme is shown separately in the upper part of FIG. 42 and the second scheme is shown separately in the lower part.
  • the first scheme shows a first example of the structural analysis method of the present invention
  • the second scheme shows a second example of the structural analysis method of the present invention.
  • the wording in the figure is an example for convenience of explanation, and does not limit the present invention.
  • a coated crystal 102 placed on a plate 101 is prepared.
  • the coated crystal 102 is coated with a gel and is a coated crystal manufactured by the crystal manufacturing method of the present invention.
  • the coated crystal is immersed in an anti-freezing agent and further scooped with a loop-shaped mounting device as described above.
  • the coated crystal thus picked up is frozen by a low-temperature gas spraying device or the like (freezing step), and the frozen crystal is subjected to structural analysis by X-ray crystal structure analysis or neutron beam crystal structure analysis.
  • the first example of the structural analysis method of the present invention can be implemented.
  • the coated crystal can be skimmed and frozen using the loop-shaped mounting device manually.
  • the loop-shaped mounting device may be referred to as “cryo loop”, for example.
  • a coated crystal 102 and a tube 103 are prepared.
  • the coated crystal 102 is coated with a gel and is a coated crystal manufactured by the crystal manufacturing method of the present invention.
  • the tube 103 may be provided with a holding portion for holding the tube 103 itself with another instrument or device at the base thereof.
  • the tip of the tube 103 is pierced into the coated crystal 102, and the portion containing the crystal is taken into the tip of the tube 103 together with the coated gel.
  • only the crystals and gel taken in at the tip of the tube 103 are separated from other parts of the coated crystal 102 by, for example, laser processing.
  • the tip of the tube 103 in which the crystals and gel are taken is immersed in an anti-freezing agent prepared in a separate container.
  • the tube 103 is placed with the tip facing upward (mount), and the coated crystal taken therein is frozen by a low-temperature gas spraying device or the like (freezing step), and the frozen crystal is analyzed by X-ray crystal structure analysis.
  • structural analysis is performed by neutron beam crystal structure analysis or the like.
  • This second example can be performed, for example, as an “automount” method in which the above steps are automated as necessary.
  • the coated crystal 102 may be prepared inside another tube.
  • the tip of the tube 103 may be pierced into the other tube, and the portion containing the crystal may be taken into the tip of the tube 103 together with the coated gel.
  • the subsequent steps can be performed in the same manner as in the second scheme of FIG.
  • the removal method is not particularly limited.
  • a method of cooling a thermoreversible hydrogel there are a method of processing with a laser beam such as a femtosecond laser, and the like.
  • the crystallization screening method of the present invention is a crystallization screening method for screening a crystallization condition of a biological material, the crystal manufacturing step for manufacturing the biological material crystal, and the crystal manufacturing step. Including the screening step of screening for crystallization conditions in the method, wherein the crystal is produced by the crystal production method of the present invention or the frozen crystal production method of the present invention.
  • the crystallization screening method of the present invention since the crystal can be easily manufactured by the crystal manufacturing method of the present invention, it is not necessary to set the crystal manufacturing conditions so strictly, the screening is also easy. It can be carried out. Specifically, for example, optimal conditions such as the concentration of the biological material and the concentration ratio (mixing ratio) of the biological material and the precipitating agent can be easily screened. Moreover, since the frozen crystal can be easily manufactured by the method for manufacturing a frozen crystal of the present invention, screening of the frozen crystal manufacturing condition can be easily performed. More specifically, for example, as described above, the crystal damage caused by the cryoprotectant can be greatly suppressed, so that screening of conditions such as the cryoprotectant concentration can be simplified.
  • the crystallization screening apparatus of the present invention is a crystallization screening apparatus for screening a crystallization condition of a biological material, the crystal manufacturing means for manufacturing the biological material crystal, and the biological material crystal. Screening means for screening crystallization conditions, wherein the crystal production means includes crystallization means for crystallizing the biological material in a gel.
  • the crystallization screening apparatus of the present invention is not particularly limited.
  • the configuration can be simplified as compared with the conventional crystallization screening apparatus.
  • it is possible to simplify the screening of conditions such as the concentration of the biological material, the concentration ratio of the biological material and the precipitating agent (mixing ratio), the concentration of the cryoprotectant, etc. Simplification is also possible.
  • the crystallization screening method of the present invention may be performed using any apparatus or instrument, but is preferably performed by the crystallization screening apparatus of the present invention.
  • FIG. 44 shows three examples of the crystallization screening apparatus of the present invention and the crystallization screening method of the present invention using the same.
  • Three examples of the crystallization screening method are referred to as a hanging drop method, a sitting drop method, and a concentration gradient method, respectively.
  • the upper left of FIG. 44 is an example of an apparatus used for the hanging drop method
  • the lower left is an example of an apparatus used for the sitting drop method
  • the upper right is an example of an apparatus used for the concentration gradient method.
  • this apparatus includes a lid 201 and a plate 203 as main components. These constitute the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serve as the screening means.
  • the lid 201 has a plate shape, and a projection 202 is provided on one surface thereof, and the other surface has a flat shape.
  • the upper surface of the protrusion 202 has, for example, a flat shape so that a gel can be placed thereon.
  • a well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 203.
  • a projection 202 formed on the lid 201 can be fitted into the well.
  • the number of wells formed on the protrusion 202 provided on the lid 201 and the plate 203 is arbitrary, and one or more wells may be provided, but a sufficient number is provided for efficient screening. Is preferred.
  • the protrusion 202 be removable from the lid 201 main body, for example, because an operation such as collecting a crystal can be easily performed.
  • the material for forming each component is not particularly limited.
  • a material that is generally used for physics and chemistry equipment, such as glass and plastic, and that does not interfere with crystal production and screening may be used.
  • the size of each component is not particularly limited, but for example, it is preferable that the entire device is of a size convenient for handling on an experimental table.
  • the crystallization screening method (hanging drop method) of the present invention using this apparatus can be performed as follows, for example. That is, first, the lid 201 is prepared with the protrusion 202 facing upward. Next, a gel containing a biological material is placed on the upper surface of the protruding portion 202, or is placed on the upper surface of the protruding portion 202 as a solution containing a biological material and solidified (gelled) there.
  • the method for producing this gel is as described above, for example. In the figure, “a mixture of protein and gel” is described. However, the present invention is not limited to protein, and any biological substance may be used.
  • a precipitating agent (precipitating agent) solution is placed in the well of the plate 203.
  • the method for producing the precipitating agent solution is also as described above, for example.
  • the lid 201 is turned upside down so that the projection 202 faces downward, and the projection 202 is fitted into the plate well. Accordingly, the gel containing the biological material is immersed in the precipitant (precipitating agent) solution, and the biological material is crystallized in the gel.
  • the crystal manufacturing process in the crystallization screening method of the present invention can be performed. In this crystal manufacturing process, various conditions such as the biological substance concentration and the precipitating agent concentration are changed, and after the crystal manufacturing process, the crystal formation state under each condition is observed. Thus, the screening step of screening for crystallization conditions is performed.
  • the screening can be performed in a single crystal manufacturing process by variously changing the biological material concentration and the precipitating agent concentration in each protrusion 202 and well. It is also possible to carry out the process. As described above, the crystallization screening method (hanging drop method) of the present invention using this apparatus can be carried out.
  • this apparatus has a plate 207 as a main component.
  • the plate 207 constitutes the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serves as the screening means.
  • a well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 207.
  • the number of the wells is arbitrary and may be one or more, but it is preferable to provide a sufficient number for efficient screening. Note that the forming material and the size of this device are the same as those in the upper left of FIG.
  • the crystallization screening method (sitting drop method) of the present invention using this apparatus can be performed, for example, as follows. That is, first, a gel containing a biological material is placed in the well of the plate 207 or placed in the well in the state of a biological material solution and solidified (gelled) there. The method for producing this gel is as described above, for example. Although it is described as “a mixture of protein and gel” in the drawing, it is not limited to protein, and any biological material may be used. Next, a precipitant (precipitant) solution is injected into the well. The method for producing the precipitating agent solution is also as described above, for example.
  • the gel containing the biological material is immersed in the precipitant (precipitating agent) solution, and the biological material is crystallized in the gel.
  • a seal or the like is preferably provided on the upper surface of the plate 207 in order to prevent foreign substances from entering the well, volatilization of the precipitant solution, and the like.
  • the screening step can be performed in a single crystal manufacturing step by variously changing the biological material concentration and the precipitating agent concentration in each well. is there.
  • the crystallization screening method (sitting drop method) of the present invention using this apparatus can be carried out.
  • this apparatus has a lid 209 and a plate 206 as main components. These constitute the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serve as the screening means.
  • the lid 209 has a plate shape, and a part of one surface thereof protrudes to form a tube 205.
  • the inside of the tube 205 is hollow, and the end on the side in contact with the lid 209 main body is open.
  • the other end of the tube 205 (hereinafter referred to as “protruding end”) is closed, and, for example, a semipermeable membrane (dialysis membrane) or the like is stretched, so that solid matter does not permeate, and only particles having a certain size or less. It can be transmitted.
  • a well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 206.
  • a tube 205 formed on the lid 209 can be fitted into the well.
  • the number of wells formed on the tube 205 and the plate 206 formed on the lid 209 is arbitrary, and may be one or more, but it is preferable to provide a sufficient number for efficient screening.
  • the forming material and the size of this device are the same as those in the upper left of FIG. 44 except for the semipermeable membrane (dialysis membrane) at the end of the tube 205.
  • the crystallization screening method (concentration gradient method) of the present invention using this apparatus can be performed, for example, as follows. That is, first, a gel containing a biological material is placed inside the tube 205 formed on the lid 209. The method for producing this gel is as described above, for example. The gel may be injected into the tube 205 after being in a gel state, but it is preferable that the gel is injected into the tube 205 in a solution (sol) state and gelled in the tube 205. In the figure, “a mixture of protein and gel” is described. However, the present invention is not limited to protein, and any biological substance may be used. On the other hand, a precipitant (precipitant) solution is placed in the well of the plate 206.
  • the method for producing the precipitating agent solution is also as described above, for example.
  • the tube 205 is fitted into the well of the plate 206.
  • the precipitating agent penetrates into the gel through the tip of the tube 205.
  • the gel 205 does not permeate due to a semipermeable membrane (dialysis membrane) or the like stretched around the protruding end of the tube 205.
  • a seal or the like is preferably installed on the upper surface of the lid 209 in order to prevent foreign matter from entering the well, drying of the gel, and the like. Thereby, the crystal manufacturing process in the crystallization screening method of the present invention can be performed.
  • the precipitating agent penetrates into the gel through the tip of the tube 205, a gradient is formed in the concentration of the precipitating agent (precipitating agent) in the gel. That is, the concentration of the precipitating agent increases on the side closer to the tip of the tube 205, and the concentration of the precipitating agent decreases as the distance from the tip increases.
  • the concentration gradient By forming this concentration gradient, the ratio between the concentration of the biological material and the concentration of the precipitating agent varies in various parts inside the tube 205. For this reason, after the completion of the crystal production process, the screening process for screening the crystallization conditions can be performed by observing the crystal formation state in the tube 205.
  • the screening step can be performed in more detail by changing the biological material concentration and the precipitant concentration in each tube 205 and well. It is.
  • the crystallization screening method (concentration gradient method) of the present invention using this apparatus can be carried out.
  • the crystallization screening method by the concentration gradient method can also be performed using a centrifuge, for example, as in Examples described later.
  • Various conditions such as the types and concentrations of the biological material and the gelling agent are not limited to the examples described later, and can be arbitrarily set. Specifically, it is the same as that of other embodiment, for example.
  • the centrifugation speed and the like are not particularly limited and can be set as appropriate.
  • the crystallization screening method by the concentration gradient method can be performed not only by forming a precipitant concentration gradient but also by forming a biological material concentration gradient, for example.
  • a gel containing no biological material is prepared in advance, and the biological material solution is gradually permeated from one side of the gel to form a gradient of the biological material concentration in the gel. You may do it.
  • the gel may or may not contain a precipitating agent in advance, for example.
  • the crystallization screening apparatus and the crystallization screening method of the present invention are not limited to these, and any apparatus and method may be used.
  • various conditions of the crystallization screening method are not limited to the above description, and can be changed as appropriate.
  • FIG. 44 as a crystallization method, a method of bringing the gel containing the biological material solution into contact with or in proximity to the precipitant solution is shown.
  • the crystallization method using a precipitating agent is, for example, mixing a precipitating agent together with a gelling agent in the biological material solution and gelling it to obtain a gel containing the precipitating agent.
  • the crystal may be precipitated by allowing it to stand as it is.
  • the crystallization screening apparatus for example, as shown in FIG. 45, an apparatus in which the tube 205 of the “concentration gradient method” apparatus (apparatus shown in the upper right of FIG. 44) is replaced with a shallow well 305 may be used. 45, the lid 304 and the plate 306 are the same as the “concentration gradient method” apparatus (the apparatus shown in the upper right of FIG. 44) except that the depth of the well is shallow to match the well 305.
  • the crystallization screening method using this apparatus is the same as the apparatus of the “concentration gradient method”, except that there is no concentration gradient formation in the gel, or is the same as the other apparatus shown in FIG.
  • 45 has an advantage that the overall operation is simpler than the apparatus of the “concentration gradient method” because the wells formed in the lid 304 and the plate 306 are shallow. 45, since the gel and the precipitating agent solution are present in different wells, the gel and the precipitation are compared with the devices of the “hanging drop method” and the “sitting drop method”. There is an advantage that the separation operation from the agent solution is simple.
  • the crystallization screening method of the present invention is performed automatically, for example.
  • the crystal manufacturing process may be automated by a method of automatically dispensing the solution (sol) of the biological material into each well or the inside of a tube by a dispensing device.
  • a dispensing device with a temperature controller
  • the biological material is not limited to proteins, and any material may be used. Any dispensing device may be used.
  • the screening process may be automated by automatically observing and photographing the crystal formation state with a microscope as illustrated.
  • the crystallization screening apparatus of the present invention includes a crystal manufacturing means for manufacturing a crystal of biological material, and therefore can be used as a crystal manufacturing apparatus for manufacturing a crystal of biological material.
  • Example 1 to 7 and Comparative Examples 1 to 7 below protein crystals of the following (1) to (3) were used as specimens (analyzed substances).
  • the following (1) to (2) and (4) to (7) protein crystals or (8) nucleic acid (DNA) crystals were used as specimens (analytes).
  • (1) Lysozyme (2) Glucose isomerase (3) Saumatine (4) Elastase (5) Synechococcus-derived phosphoribrokinase (PRK) (Phosphoribokinase (PRK) / Synechococcus) (6) Serine acetyltransferase (SAT) (7) E. coli foreign body excretion transporter (AcrB) (8) DNA
  • the trade name Ultrax-18 (Cu counter cathode) manufactured by Rigaku Corporation was used as the X-ray source.
  • the detector used was R-AXIS IV ++ manufactured by Rigaku Corporation.
  • the measurement voltage was 50 kV
  • the measurement current was 100 mA
  • the beam diameter was 0.3 mm.
  • Example 1 Agarose-III (manufactured by Dojindo Laboratories Co., Ltd., gelation temperature is about 37-39 ° C.) 2% by mass and 98% by mass of water dissolved by heating (A) 0.1 mL and chicken egg white lysozyme solution (B ) 0.1 mL of both were mixed at a constant temperature of 35 ° C. to obtain a crystallization solution (C). Before the crystallization solution (C) became a solidified gel, it was filled into a capillary (manufactured by Hirgenberg).
  • the capillary was made of glass (80 mm, inner diameter 0.7 mm), and filled with a crystallization solution (C) before solidifying into an internal tubular space.
  • the crystallization solution (C) solidified after several tens of seconds at room temperature, and the capillary inner space was filled with the gel in which the crystallization solution (C) was solidified.
  • the filled capillary was then inserted into a test tube container into which approximately 2 ml of the precipitant solution was injected.
  • This test tube container was made of glass having a diameter of about 16 mm and a length of about 133 mm. At this time, a contact interface between the solidified (gelled) crystallization solution (C) and the precipitating agent solution was formed at the insertion end of the capillary.
  • the open end of the test tube was sealed with a stopper.
  • the composition of the protein solution and the precipitating agent solution is shown below.
  • Precipitating agent solution 1.0M sodium chloride 0.1M acetate buffer pH 4.5
  • Crystallization temperature 20 ° C
  • lysozyme crystals were confirmed in the solidified gel within a few days.
  • the capillary was taken out from the test tube, and the lysozyme crystal crystallized in the solidified gel in the capillary was taken out by the following method. That is, first, a glass plate was prepared, and about 30 ⁇ L each of a precipitating agent solution and an anti-freezing agent solution (100% dimethyl sulfoxide solution in this example) were dispensed on different positions. .
  • the capillary was cut or crushed within a range of 5 to 10 mm before and after the crystal to be taken out using tweezers or a capillary cutter, and then the solidified gel was cut with a cutter or the like. Furthermore, the crystals in the taken-out gel were moved into the precipitating agent solution prepared in advance. Using a micro tool (manufactured by Hampton Research) or the like, the gel around the lysozyme crystal was carefully peeled off to a thickness of about 0.1 mm under a microscope.
  • FIG. 1A and FIG. 1B show photographs of the lysozyme crystals of Example 1 protected with a gel, taken at different angles.
  • Example 1 a large and high quality lysozyme single crystal was obtained.
  • 2A and 2B show X-ray diffraction images of the lysozyme crystal of Example 1.
  • FIG. FIG. 2A is an overall view
  • FIG. 2B is an enlarged view of a part (the frame line portion at the lower right of FIG. 2A). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • Example 2 A glucose isomerase solution was used in place of the chicken egg white lysozyme solution, and crystals were produced in the same manner as in Example 1 except for the composition of the protein solution and the precipitating agent solution and the type of the cryoprotectant. About 1 week after setup at a crystallization temperature of 4 ° C., glucose isomerase crystals were confirmed in the solidified gel. The composition of the protein solution and the precipitating agent solution is shown below.
  • Protein solution 25 mg / mL glucose isomerase 0.1 M Hepes buffer pH 7.5 1 mM magnesium chloride
  • Precipitating agent solution 2.5M ammonium sulfate 0.1M Hepes buffer pH7.5 Crystallization temperature: 4 °C
  • the antifreezing agent solution was subjected to X-ray crystal structure analysis using a 2.5M lithium acetate solution.
  • the glucose isomerase crystal could be easily captured without damage, and it was resistant to liquid nitrogen treatment without being damaged by the antifreezing agent solution, and a good X-ray diffraction image was obtained.
  • FIG. 3A, 3B, 4, 5A and 5B show X-ray diffraction images of the glucose isomerase crystal of Example 2.
  • FIG. 3A is an overall view
  • FIG. 3B is an enlarged view of a part (the frame line portion at the upper right of FIG. 3A).
  • FIG. 4 is an X-ray diffraction image taken under different conditions.
  • FIG. 5A is an overall view of an X-ray diffraction image measured by further changing the conditions
  • FIG. 5B is an enlarged view of a part (the frame line part at the lower right of FIG. 5A). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • 6A to 6D show the lysozyme crystal of Example 2.
  • FIG. 1 is an overall view
  • FIG. 3B is an enlarged view of a part (the frame line portion at the upper right of FIG. 3A).
  • FIG. 4 is an X-ray diffraction image taken under
  • FIG. 6A is a diagram showing a state immediately before dipping in the antifreezing agent solution, and a single crystal is obtained.
  • FIG. 6B shows a state after 5 seconds from the immersion in the cryoprotectant solution
  • FIG. 6C shows a state after 1 minute from the immersion
  • FIG. 6D shows a state after 5 minutes after the immersion.
  • the crystals on the right side of FIGS. A to D collapsed gradually after immersion because they had some defects before immersion in the cryoprotectant solution, but the crystals on the left side were good single crystals and were completely disintegrated even after being immersed for 5 minutes. I did not.
  • Example 5 Crystallization was carried out in the same manner as in Example 1 except that a thaumatin solution was used in place of the chicken egg white lysozyme solution and the composition of the protein solution and the precipitating agent solution and the type of the antifreezing agent were used. After setting up at a crystallization temperature of 20 ° C., glucose isomerase crystals were confirmed in the gel solidified in about one week. Below, the composition of a protein solution and a precipitant solution is shown.
  • Protein solution 20 mg / mL thaumatin 0.1 M N- (2-acetamido) iminodiacetic acid buffer pH 6.5
  • Precipitating agent solution 2.0 M potassium sodium tartrate 0.1 M N- (2-acetamido) iminodiacetic acid buffer pH 6.5
  • the cryoprotectant solution was subjected to X-ray crystal structure analysis using 100% glycerol.
  • thaumatin crystals could be easily captured without damage, and withstand damage to liquid nitrogen without damage caused by the antifreezing agent solution, and a good X-ray diffraction image was obtained.
  • FIG. 7A shows a photograph of the thaumatin crystal of Example 5 protected with a gel. As illustrated, in Example 5, a large and high quality thaumatin single crystal was obtained. 7B and 7C show X-ray diffraction images of the thaumatin crystals of Example 5. FIG. FIG. 7B is an overall view, and FIG. 7C is an enlarged view of a part (the frame line portion at the bottom of FIG. 7B). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • FIG. 8A shows a photograph of the thaumatin crystal of Example 7 protected with a gel. As illustrated, in Example 7, a large and high quality thaumatin single crystal was obtained. 8B and 8C show X-ray diffraction images of the thaumatin crystal of Example 7. FIG. FIG. 8B is an overall view, and FIG. 8C is an enlarged view of a part (a frame portion at the bottom of FIG. 8B). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • agarose-III manufactured by Dojindo Laboratories, Inc., gelation temperature is about 37 to 39 ° C.
  • agarose IX-A manufactured by Sigma, gelation temperature is about 8 to 17 ° C.
  • crystals and frozen crystals were produced in the same manner as in Examples 1 to 7 except that the temperature conditions were changed, and similarly good results were obtained.
  • Example 1 Crystallization was carried out in exactly the same manner as in Example 1, except that a lysozyme solution without addition of agarose was used. After setup, lysozyme crystals were confirmed within a few days. The lysozyme crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the antifreeze solution (100% dimethyl sulfoxide solution). Therefore, X-ray crystal structure analysis could not be performed.
  • 9A and 9B show photographs of the lysozyme crystal of Comparative Example 1.
  • FIG. FIG. 9A is a photograph showing a state immediately before dipping in the cryoprotectant solution, and a single crystal is obtained.
  • FIG. 9B is a photograph showing a state immediately after being immersed in the anti-freezing agent solution, and the crystals are shattered.
  • Example 2 Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (2.5M lithium acetate solution). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 3 Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (100% glycerol). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 4 Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystal taken out from the capillary was broken into pieces and melted at the moment of immersion in the antifreeze solution (50% glycerol solution). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 5 Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (100% glycerol). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 6 Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and dissolved at the moment of immersion in an anti-freezing agent solution (100% polyethylene glycol (average molecular weight 400)). Therefore, X-ray crystal structure analysis could not be performed. 10A and 10B show photographs of the lysozyme crystal of Comparative Example 6. FIG. FIG. 10A is a photograph showing a state immediately before dipping in an anti-freezing agent solution, and a single crystal is obtained. FIG. 10B is a photograph showing a state immediately after being immersed in the cryoprotectant solution, and the crystals are shattered.
  • Example 7 Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in an anti-freezing agent solution (60% polyethylene glycol (average molecular weight 4000) solution). Therefore, X-ray crystal structure analysis could not be performed.
  • an anti-freezing agent solution 60% polyethylene glycol (average molecular weight 4000) solution. Therefore, X-ray crystal structure analysis could not be performed.
  • Example 8 A coated crystal coated with a gel was produced, and an X-ray crystal structure analysis was performed using a 2.5 M aqueous lithium acetate solution as an antifreezing agent. Using a 2.5M aqueous lithium acetate solution as an antifreezing agent, setting the immersion time of the coated crystal in the antifreezing agent to 15 minutes, the biological material constituting the crystal to be analyzed, and the gel (Agarose III) The same procedure as in Example 1 was performed except for the concentration.
  • Example 8 is one in which elastase, which is a kind of protein, is used as the biological material, and the gel (agarose III) concentration is 2.0 mass%.
  • Example 9 uses lysozyme which is a kind of protein as the biological material, and the concentration of gel (agarose III) is 1.6% by mass.
  • Example 10 uses glucose isomerase, which is a kind of protein, as the biological material and the gel (agarose III) concentration is 1.6 mass%.
  • Example 8 is one in which elastase which is a kind of protein is used as the biological material, and the immersion time in the cryoprotectant is 2 seconds.
  • Example 9 uses lysozyme which is a kind of protein as the biological material, and the immersion time in the cryoprotectant is 15 seconds.
  • FIG. 11 shows photographs and data of the crystals of Examples 8 to 10 and Comparative Examples 8 to 9. It is a photograph of comparative example 8, example 8, comparative example 9, example 9, and example 10, respectively from left to right.
  • the numerical value in the lower part of the figure shows the amount of agarose added (mass%), the immersion time in the cryoprotectant (Cryo) solution, and the mosaic property in the X-ray crystal structure analysis.
  • the coated crystals of Examples 8 to 10 were not damaged even when immersed for a long time of 15 minutes in an anti-freezing agent (2.5 M lithium acetate aqueous solution).
  • the mosaic property of these crystals is a sufficiently small value of about 0.3, indicating that the crystals are of high quality.
  • the crystal of Comparative Example 8 had a large mosaic property of 1.17, and the crystal of Comparative Example 10 was damaged when immersed in an anti-freezing agent for 15 seconds, and X-ray crystal structure analysis could not be performed.
  • Example 11 Coated crystal production and X-ray crystal structure analysis were performed under the same conditions as in Example 10 except that the immersion time in the antifreezing agent (2.5 M lithium acetate aqueous solution) was changed to 10 minutes. As a result, the mosaic value is 0.08 ° including the beam dispersion, which is an extremely small value, indicating that the crystal has a very high quality over the prior art.
  • FIG. 12 shows an X-ray diffraction image of the crystal of this example. The resolution at this time was 0.93 mm, and the crystal to be analyzed was a large crystal having a diameter of 0.5 mm or more.
  • Example 12 Production of crystals under conditions of changing gel concentration and screening of crystallization conditions
  • the agarose-III concentration in the agarose-III solution (A) was variously changed from 0 to 2.0% by mass in increments of 0.2% by mass, and the lysozyme concentration in the protein solution (B) was changed from 30 mg / mL.
  • a coated crystal coated with agarose-III gel was produced in the same manner as in Example 1 except that it was carried out by the batch method using Imp @ ct plate (trade name) manufactured by Hampton Research Co., Ltd.
  • the crystallization conditions (crystal production conditions) under the changing conditions were screened.
  • description will be made using the reference numerals in the lower left of FIG. 44 (sitting drop method). Since this figure is a schematic diagram, the number of wells, the dimensional ratio of each part, etc. It is different from plate. That is, first, the agarose-III concentration in the agarose-III solution (A) was prepared at various agarose-III concentrations changed from 0 to 2.0% by mass in steps of 0.2% by mass as described above. This was mixed with the protein solution (B) to prepare crystallization solutions (C) having different agarose-III concentrations.
  • the crystallization solution (C) having different agarose-III concentrations was mixed with the precipitating agent solution, and 6 ⁇ L was injected into each well of the crystallization plate 207.
  • a total of eight crystallized samples with the same agarose-III concentration were prepared and injected into separate wells.
  • This crystallization solution (C) formed droplets in the well and immediately gelled.
  • a seal was affixed to the upper surface of the plate (crystallization plate) 207 to prevent the gel in each well from being exposed to the atmosphere, and left to stand at 20 ° C. for 3 days. Thereafter, the number of crystals formed in each sample was observed with a microscope. The number of produced crystals of eight samples at each concentration was averaged to obtain the average number of crystals (pieces).
  • the horizontal axis represents the agarose-III concentration
  • the vertical axis represents the average number of crystals (pieces).
  • Example 13 Crystal production and crystallization condition screening under gel concentration changing conditions
  • the type of agarose was agarose-III (Agarose III), agarose 9A, or agarose Sea Plaque (agarose SP), and agarose SeaKem (all are trade names of Takara Bio Inc.), and the agarose concentration was 0, 0. .2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5
  • the gel was changed in the same manner as in Example 12 except that it was variously changed to 4.0, 4.5, 5.0, 5.5, or 6.0% by mass (for SeaKem up to 2.0% by mass or less).
  • the crystallization conditions (crystal production conditions) under the concentration change conditions were screened.
  • the graph of FIG. 16 shows the results when the agarose concentration is 0 to 2.0% by mass.
  • the graph on the upper left of the figure is a graph showing the results of agarose 9A.
  • the graph in the upper right of the figure is a graph showing the results of agarose SP.
  • the graph on the lower left of the figure is a graph showing the results of agarose-III.
  • the graph on the lower right of the figure is a graph showing the results of agarose SeaKem.
  • the horizontal axis represents the agarose concentration
  • the vertical axis represents the average number of crystals (pieces). Further, the graph of FIG.
  • the graph 17 shows the results when the agarose concentration is 0 and 2.0 to 6.0 mass%.
  • the graph in the upper right of the figure is a graph showing the results of agarose-III.
  • the graph on the lower left of the figure is a graph showing the results of agarose 9A.
  • the lower right graph is a graph showing the results of agarose SP.
  • the horizontal axis represents the agarose concentration
  • the vertical axis represents the average number of crystals (pieces).
  • the mechanism of the correlation between the agarose concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (concentration other than 0), it was shown that good crystal production conditions were obtained. It was done.
  • Example 14 Crystal production under crystallization condition change and crystallization condition screening
  • the type of agarose is NuSieve 3: 1 (trade name of Takara Bio Inc.), and the agarose concentration is 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 mass%
  • the crystallization conditions (crystal production conditions) under gel concentration change conditions were screened in the same manner as in Example 12 except that various changes were made. The result is shown in the graph of FIG. In the figure, the horizontal axis represents NuSieve 3: 1 concentration, and the vertical axis represents the average number of crystals (pieces).
  • Example 15 Crystal production and crystallization condition screening under changing conditions of gel concentration and precipitant concentration
  • the crystallization conditions (crystal production conditions) under the conditions of changing the gel concentration and the precipitating agent were screened in the same manner as in Example 12 except that the immersion time was 6 days or 7 days.
  • the gel concentration was changed from 0 to 2.0% by mass in increments of 0.2% by mass for each sodium chloride concentration as in Example 12.
  • the result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces).
  • Example 16 Crystal production and crystallization condition screening under conditions of changing gel concentration and precipitating agent concentration
  • high melting point agarose-III was used instead of the normal agarose-III used in each of the above Examples, the crystallization conditions (crystal production under the conditions of changing gel concentration and precipitating agent) Condition).
  • the gel concentration was changed from 0 to 2.0% by mass in increments of 0.2% by mass for each sodium chloride concentration as in Example 15.
  • the result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces).
  • Example 17 Crystal production under crystallization condition change and crystallization condition screening
  • agarose-III concentration 0 or 2.0
  • elastase was used instead of lysozyme as the protein
  • concentration of elastase in the protein solution (B) was changed.
  • Example 12 except that it was 12.5 mg / mL, that the type of agarose was agarose seaplaque (SP), and that the number of samples used to calculate the average number of crystals was 12 instead of 8.
  • crystallization conditions crystal production conditions under gel concentration changing conditions were screened. The result is shown in the graph of FIG.
  • the horizontal axis represents the agarose SP concentration
  • the vertical axis represents the average number of crystals (pieces).
  • Example 18 Crystal production under crystallization condition change and crystallization condition screening
  • Glucose isomerase was used instead of lysozyme as the protein
  • the concentration of glucose isomerase was 10 mg / mL in the protein solution (B)
  • the type of agarose was ultra-low molecular weight agarose 9A
  • the precipitating agent was chlorinated.
  • a 0.1 M calcium chloride (CaCl 2 ) aqueous solution containing 10% by mass of 2-methyl-2,4-pentanediol (MPD) was used.
  • Example 19 Crystal production under crystallization condition change and crystallization condition screening
  • the type of agarose being agarose SP and the precipitating agent being a 0.1 M calcium acetate (CaAc) aqueous solution containing 8.0% by mass of 2-methyl-2,4-pentanediol (MPD).
  • crystallization conditions under gel concentration change conditions were screened.
  • the results are shown in the photograph and graph of FIG.
  • the numerical value at the bottom of each photograph indicates the agarose SP concentration (% by mass).
  • the horizontal axis represents the agarose SP concentration
  • the vertical axis represents the average number of crystals (pieces).
  • the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated.
  • Example 20 Crystal production and crystallization condition screening under changing conditions of protein concentration and gel concentration
  • the horizontal axis represents the agarose SP (Seaplaque) concentration
  • the vertical axis represents the average number of crystals (pieces).
  • the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated. Moreover, it was shown that good crystal production conditions were obtained regardless of whether the thaumatin concentration was 50 mg / mL or 25 mg / mL.
  • Example 21 Crystal production under crystallization condition change and crystallization condition screening
  • Crystal production and crystallization condition screening were performed in the same manner as in Example 12 except for the following (1) to (3).
  • (1) Crystals were produced by the hanging drop method using VDX plate (trade name of Hampton Research).
  • a mixed solution with 2 HgCl was used as a crystallization solution (C).
  • this crystallization solution (C) gelatinized immediately, it used for the crystal manufacturing process immediately after preparation.
  • the crystal manufacturing process by the hanging drop method was performed as follows. That is, first, 500 ⁇ L / well of the precipitant solution was prepared in advance in each well (plate well) of the plate 203. On the other hand, 2 ⁇ L of the crystallization solution (C) prepared as described above was dispensed on the protrusion 202 (cover glass) to be gelled. For each concentration of the crystallization solution (C), eight identical samples were placed on top of the eight protrusions 202. Thereafter, the lid 201 was turned upside down so that the projections 202 faced down, and the projections 202 were fitted into the plate well containing the precipitating agent solution to bring the gel and the precipitating agent solution close to each other.
  • Example 22 Crystal production of E. coli-derived protein (crystallization)
  • Escherichia coli-derived serine acetyltransferase (SAT) 7.5 mg / mL was used instead of the above protein, and a precipitating agent (precipitating agent) solution was added to 4.5% by mass of PEG 8000 (Promega's polyethylene glycol) ) And 0.1M sodium cacodylate in pH 6.5 aqueous solution, except that the crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened in the same manner as in Example 21.
  • SAT Escherichia coli-derived serine acetyltransferase
  • Example 23 Crystal production of E. coli-derived protein (crystallization)] Same as Example 22 except that the precipitating agent (precipitating agent) solution was a pH 6.5 aqueous solution containing 7.5% by mass of PEG8000 (trade name of polyethylene glycol from Promega) and 0.1 M sodium cacodylate. Thus, crystallization conditions (crystal production conditions) under gel concentration change conditions were screened.
  • Example 24 Crystal production of E. coli-derived protein (crystallization)
  • E. coli foreign body excretion transporter (AcrB) 28 mg / mL was used instead of SAT, and a precipitating agent (precipitating agent) solution was 10% by mass of PEG2000 (trade name of polyethylene glycol of Promega), Crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened in the same manner as in Example 12 except that the aqueous solution was pH 5.6 containing 80 mM sodium dihydrogen phosphate and 20 mM sodium citrate.
  • Example 25 Crystal production of E. coli-derived protein (crystallization)
  • the crystallization conditions under the gel concentration changing conditions were the same. Screened.
  • Example 26 Crystal production of nucleic acid (DNA) and screening of crystallization conditions
  • a crystal of nucleic acid (DNA) was produced as follows.
  • DNA was annealed. That is, first, a 1 mM aqueous solution of DNA1 (5′-AAGAAAAAAA-3 ′: SEQ ID NO: 1), which is a single-stranded DNA, and DNA2 (5′-TTTTTTTCTT-3 ′: SEQ ID NO: 2), which is a complementary strand of DNA1, are used. An equal amount of 1 mM aqueous solution was mixed, and left at 60 ° C. for 10 minutes and then at 20 ° C. for 15 minutes. Here, 1 mM aqueous solution of BNA (Bridged Nucleic Acid, Gene Design Co., Ltd.) was added in the same amount as the DNA1 aqueous solution, and allowed to stand at 20 ° C. overnight. In this way, DNA annealing was performed to obtain an annealed DNA aqueous solution. This annealed DNA aqueous solution was used for crystal production.
  • DNA1 5′-AAGAAAAAAA-3 ′: SEQ ID NO: 1
  • the crystallization solution (C) (mixed aqueous solution of gel and DNA) was prepared as follows. That is, first, agarose 9A was dissolved in water at 85 ° C., and an aqueous gelling agent solution (4 times the final concentration) kept at 37 ° C. was prepared. This gelling agent aqueous solution, a buffer containing MPD, sodium cacodylate and MgCl 2 at a concentration four times the final concentration, and the annealed DNA aqueous solution are mixed in this order at a volume ratio of 1: 1: 2. A crystallization solution (C) was obtained. Since this crystallization solution (C) was gelated immediately, it was subjected to the following crystal production process immediately after preparation.
  • the crystal manufacturing process was performed as follows. That is, first, 500 ⁇ L / well of the precipitant solution was prepared in advance in each well (plate well) of the plate 203. On the other hand, 2 ⁇ L of the crystallization solution (C) prepared as described above was dispensed on the protrusion 202 (cover glass) to be gelled. For each concentration of the crystallization solution (C), eight identical samples were placed on top of the eight protrusions 202. Thereafter, the lid 201 was turned upside down so that the projections 202 faced down, and the projections 202 were fitted into the plate well containing the precipitating agent solution to bring the gel and the precipitating agent solution close to each other. After setting in this way, the mixture was allowed to stand at 20 ° C. for 1 to 5 days, and DNA crystals were precipitated in the gel to produce crystals.
  • FIG. 27 shows a process of producing a DNA crystal (crystal growth) in this example.
  • the left column of FIG. 27 is an example in which agarose was not added (corresponding to a comparative example).
  • the middle column is an example with an agarose 9A final concentration of 1.0 mass%.
  • the right column is an example of a final concentration of 1.6% by mass of agarose 9A.
  • the upper stage is a photograph immediately after standing at 20 ° C. for 1 day
  • the lower stage is a photograph immediately after standing at 20 ° C. for 5 days.
  • the DNA crystal grows without adding agarose, but according to the example of the present invention to which agarose was added, it was shown that the crystal was more likely to grow.
  • the drawing when 5 days passed after standing at 20 ° C., the number of fine crystals decreased and the transparency of the gel increased, and the crystals grew larger than when 1 day after standing.
  • Example 27 Crystal production and crystallization screening using mebiol gel
  • agarose-III solution A
  • meviol gel solution in which the meviol gel concentration was variously changed to 0, 1, 3, and 5% by mass was used, and the concentration of lysozyme in the protein solution (B) was changed. Lysozyme crystals were produced in the same manner as in Example 12 except that the concentration was changed to 40 mg / mL, and crystallization screening was performed.
  • FIG. 28 shows the result. In the graph on the left side of the figure, the horizontal axis represents the amount of meviol gel added (mass%), and the vertical axis represents the average number of crystals (number of crystal precipitates).
  • the photograph on the right side shows the precipitated crystals when 1% by mass of mebiol gel is added and when no mebiol gel is added.
  • mebiol gel when no mebiol gel was added, almost no lysozyme crystals were precipitated, and the precipitated crystals were cracked and were not good quality crystals.
  • mebiol gel when mebiol gel was added, many high-quality crystals without cracks were obtained.
  • the amount of meviol gel added was increased to 5% by mass, the number of crystal precipitates increased rapidly. Furthermore, even when the mebiol gel was increased to 7% by mass, the same good results were obtained.
  • the above crystals are coated crystals coated with mebiol gel.
  • mebiol gel By cooling to 15 ° C. or lower, mebiol gel is liquefied (solified) and the gel coating can be removed very easily. It was. This is shown in the photograph of FIG.
  • the photograph on the left shows crystals coated with mebiol gel before cooling.
  • the photo on the right shows the crystal after cooling, with the gel coating removed.
  • Example 13 coated crystals produced using 2.0 mass% agarose 9A, 2.0 mass% agarose SP, or 2.0 mass% agarose-III were used for evaluation.
  • Example 27 a coated crystal produced using 1% by mass of meviol gel was used for evaluation. The evaluation is performed by removing the seal from the upper surface of the well (VDX plate (trade name) manufactured by Hampton Research Co., Ltd.) for the hanging drop method containing the crystal, exposing the crystal to normal temperature atmosphere, and observing the change with time under a microscope. went.
  • the leftmost “None” line shows the change with time of a crystal (comparative example) produced without adding gel.
  • the second “2.0% 9A” line from the left shows the change over time of crystals produced using 2.0 mass% agarose 9A.
  • the third “2.0% SP” line from the left shows the change over time of crystals produced using 2.0 mass% agarose SP.
  • the rightmost “2.0% III” line shows the time course of the crystals produced using 2.0 mass% agarose-III.
  • the number represented by “min” on the left side of the photograph represents the exposure time (minutes) of the crystal to the atmosphere.
  • the crystal produced without the addition of the gel started to dry at an exposure time of 10 minutes, a remarkable crack occurred at an exposure time of 16 minutes, and was already unusable, and the exposure time was 29 minutes. Then it was completely collapsed.
  • each coated crystal of the example withstood the drying for a long time of exposure time of 20 to 29 minutes.
  • FIGS. 31A and 31B show the results of evaluation of the crystal of Example 27 against dryness.
  • the “no gel addition” line in the upper part of FIG. 31A shows the change with time of the crystal (comparative example) produced without adding the gel.
  • the line of “addition of 1% mebiol” in the lower part of FIG. 31A shows the change over time of crystals produced using 1.0 mass% meviol gel.
  • the upper part of FIG. 31B shows the change with time of the crystal (comparative example) produced without adding the gel, following the upper part of FIG. 31A.
  • the lower part of FIG. 31B shows the change over time of crystals produced using 1.0 mass% meviol gel following the lower part of FIG. 31A.
  • the number represented by “min” represents the exposure time (minutes) of the crystal to the atmosphere.
  • the crystal produced without the addition of gel started to dry on the surface after exposure time of 4 minutes, began to crack after exposure time of 22 minutes, Cracks occurred and almost completely collapsed after 36 minutes of exposure.
  • the coated crystal of the example coated with mebiol gel does not cause any deterioration such as cracking and drying even at an exposure time of 36 minutes, and even a small amount of crystals have fine cracks even at an exposure time of 41 minutes. Only entered.
  • the coated crystal produced by the production method of the present invention was extremely resistant to drying and suitable for storage because it was coated with a gel.
  • Example 28 Crystal production using mebiol gel
  • a mixed solution of lysozyme concentration 35 mg / mL, sodium chloride concentration 3% by mass, sodium acetate concentration 0.2M, mebiol gel concentration 2% by mass is kept at 12 ° C. and allowed to stand at 12 ° C. for about 1 week.
  • the crystal production method of the present invention was carried out. Thereafter, the temperature of the mixed solution was raised to 20 ° C. and converted (moved) into a gel state. It was observed whether there was a change such as damage to the crystal before and after this state change (movement). The observation result is shown in the photograph of FIG. The left is a photograph of the sol state (before movement), and the right is a photograph of the gel state (after movement). As shown, no change was observed in the crystal state before and after the movement. Further, similar results were obtained even when the meviol gel concentration was changed to 3% by mass.
  • FIG. 33 shows the results of X-ray crystal structure analysis of the crystals of Examples 27 and 28 in the table and photograph of FIG. X-ray crystal structure analysis was carried out in the same manner as in the previous examples.
  • the cryoprotectant was 2.5M lithium acetate and the immersion time was 15 minutes.
  • Number 1 in the table indicates a crystal (comparative example) produced without adding mebiol gel.
  • Numbers 2 and 3 show the crystals of Example 28 (2% and 3% by weight meviol gel).
  • Numbers 4 and 5 indicate the crystals of Example 27 (2% and 5% by weight meviol gel).
  • the photograph shows an X-ray diffraction image, and the numbers 1, 2, 3, and 5 attached to the photograph respectively correspond to the numbers in the table.
  • FIG. 34 shows the result. As shown on the left side of FIG. 34, a good crystal was obtained, and as shown on the right side, a good X-ray diffraction image and a low mosaic property (0.3178) were obtained.
  • the processed part (processed crystal) cut out by processing could be taken out using tweezers, cryoloop, etc., and used for X-ray crystal structure analysis.
  • the left side of FIG. 36 is a photograph showing a state in which the processed part (processed crystal) is being taken out
  • the right part of FIG. 36 is a photograph of the processed part (processed crystal) after being taken out.
  • FIG. 37 shows the X-ray crystal structure analysis result of the processed part.
  • the left side of FIG. 37 is a photograph showing the crystal and gel of the processed part
  • the right side is the resolution and mosaic property of each part shown on the left side of the figure. As shown in the figure, it was confirmed that the crystal included in the processed part has a good structure.
  • Such laser beam processing can be performed on crystals not covered with gel. According to this laser beam processing, non-contact processing can be performed in a sealed environment. Furthermore, since the molecular bond is cut by light energy, processing can be performed without thermal diffusion to the peripheral portion. In particular, the femtosecond laser absorbs light energy only in the vicinity of, for example, a few ⁇ m near the condensing point, so that precise three-dimensional processing or the like is possible. However, when a crystal that is present in the liquid and is not coated with a gel is processed with a laser beam, the crystal is not fixed with the gel and moves, so that accurate processing may be difficult. Moreover, since the crystal is not protected by the gel, it may be damaged.
  • the crystal used for laser beam processing is a coated crystal coated with a gel (gel-coated crystal)
  • these problems can be solved.
  • the gel-coated crystal is processed using laser light, the crystal can be processed and taken out more easily than the processing by physical means, and the gel coating has an advantage that the processing is not hindered.
  • the subject of both gel coat crystal and laser beam processing can be supplemented.
  • Example 29 Production of growth crystal using coated crystal as seed crystal
  • a part of the lysozyme crystal (agarose-III concentration: 1.0% by mass) coated with the agarose gel produced in the crystallization screening of Example 12 was cut out with the above-mentioned femtosecond laser as a seed crystal.
  • This seed crystal was put in an aqueous solution containing 3% by mass of sodium chloride and 20 mg / mL of lysozyme, and allowed to stand at room temperature for 60 days to grow the crystal, and the growth crystal production method of the present invention was carried out. This is shown in the photograph of FIG.
  • the left figure is the gel-coated crystal of Example 12, the middle figure is a seed crystal (processed crystal) that is partly cut by femtosecond laser processing, and the right figure is after growth for 60 days at room temperature. It is a grown crystal. As shown in the figure, growth in a solution yielded a large crystal that was much larger than the seed crystal and had a diameter of about 1 mm. Such a large crystal can be used for various applications, and is suitable for, for example, neutron beam crystal structure analysis.
  • Example 30 Production of growth crystal using coated crystal as seed crystal
  • a part of the lysozyme crystal coated with meviol gel (meviol gel concentration: 5.0% by mass) produced in the crystallization screening of Example 27 was cut out with the femtosecond laser as a seed crystal.
  • This seed crystal was placed in an aqueous solution containing 3% by mass of sodium chloride and 20 mg / mL of lysozyme, and allowed to stand at room temperature for 4 days to grow a crystal, and the growth crystal production method of the present invention was carried out. This is shown in the photograph of FIG.
  • the left figure is the gel-coated crystal of Example 27, the middle figure is a seed crystal (processed crystal) that is partly cut by femtosecond laser processing, and the right figure is after growth for 4 days at room temperature. It is a grown crystal. As shown in the figure, growth in a solution yielded a large crystal that was much larger than the seed crystal and had a diameter of about 0.5 mm. Such a large crystal can be used for various applications as described in Example 29, and is suitable for, for example, neutron beam crystal structure analysis.
  • Example 31 Crystallization screening using concentration gradient by centrifugation
  • Crystallization screening was performed by forming a gradient in the concentration of the precipitating agent (precipitating agent) between the upper and lower parts inside the centrifuge tube using a centrifuge.
  • precipitating agent precipitating agent
  • a centrifuge tube 405 (manufactured by BD, trade name: BD Falcon Cell Culture Inserts) was prepared.
  • This centrifuge tube has a PET semipermeable membrane (dialysis membrane) attached to the bottom, and the semipermeable membrane allows only particles of a certain size or less to pass through without passing through solids.
  • a 2.0 mass% agarose aqueous solution (agarose type is Seaplaque) was placed in the centrifuge tube 405 and gelled.
  • aqueous solution containing 50 mg / mL of lysozyme, 4.0% by mass of sodium chloride, 0.1M sodium acetate, pH 4.5 was poured onto the upper surface of the gel. This was centrifuged at 20,000 rpm for 15 hours at 20 ° C. using a centrifuge (trade name Plate Spin, manufactured by Kubota Corporation), and the lysozyme solution was uniformly diffused in the gel.
  • a precipitating agent (4.0 mass% sodium chloride aqueous solution) was placed in a cylindrical container 406 that was thicker than the centrifuge tube 405 and closed at the bottom. Further, a centrifuge tube 405 containing the gel was put therein. When left at 20 ° C. for 2 days, the precipitating agent gradually permeates into the centrifuge tube 405 through the bottom PET film, and the concentration of the precipitating agent is high at the bottom and the concentration of the precipitating agent is low at the top. A lysozyme crystal was precipitated in the gel. In this way, crystals were precipitated in the gel to produce lysozyme crystals.
  • a precipitating agent 4.0 mass% sodium chloride aqueous solution
  • FIG. 41 shows the photograph. 41, the upper part of FIG. 41 shows the upper part inside the centrifuge tube 405 (cup), and the lower part of FIG. 41 shows the lower part inside the centrifuge tube 405 (cup) (PET membrane (membrane) side).
  • the number of precipitated crystals was large at the lower part of the cup (membrane side) where the precipitant concentration was high.
  • the number of precipitated crystals was small. That is, using centrifuging, a concentration gradient of the precipitating agent was formed between the upper part and the lower part inside the centrifuge container, and the crystallization conditions could be screened.
  • this Example is a screening of the crystallization conditions using the concentration gradient of a precipitating agent, the screening of the crystallization conditions by forming a protein concentration gradient can also be performed simply. The same applies to biological substances other than proteins.
  • the crystal manufacturing method of the present invention it is possible to easily manufacture a crystal of the biological substance, rather than precipitating the crystal from a solution. Moreover, according to the frozen crystal manufacturing method of the present invention, the crystal is not easily damaged by freezing. Furthermore, according to the growth crystal manufacturing method of the present invention, since the crystal is grown using the coated crystal coated with the gel as a seed crystal, a larger crystal can be obtained. In addition, the crystal of the present invention is manufactured by the crystal manufacturing method of the present invention, the frozen crystal manufacturing method of the present invention, or the grown crystal manufacturing method of the present invention, so that it is suitable for crystal structure analysis, for example. It has special characteristics. Furthermore, according to the structure analysis method of the present invention, since the structure analysis of the coated crystal coated with the gel or the frozen crystal frozen from the gel is performed, there is an advantage that the crystal is easy to handle.
  • the crystallization screening method of the present invention can easily produce crystals by the crystal production method of the present invention, it is not necessary to set the crystal production conditions so strictly, and thus screening can be performed easily. . Furthermore, the crystallization screening apparatus of the present invention can simplify the configuration of the apparatus for the same reason.
  • the crystal production method and frozen crystal production method of the present invention are useful for crystal structure analysis, particularly X-ray crystal structure analysis, and can also be applied to crystallization screening methods. Furthermore, the method for producing crystals and the method for producing frozen crystals of the present invention and the uses of the crystals and frozen crystals produced thereby are not limited to the above-mentioned uses, and can be used for all uses.

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Abstract

Disclosed is a crystal production method which is characterized in that a crystal can be produced conveniently and that a crystal produced by the method is easy to handle. Specifically disclosed is a method for producing a crystal of a biological substance, which comprises a crystallization step of crystallizing the biological substance, wherein the biological substance is crystallized in a gel in the crystallization step.

Description

結晶製造方法、凍結結晶製造方法、結晶、結晶構造解析方法、結晶化スクリーニング方法、結晶化スクリーニング装置Crystal manufacturing method, frozen crystal manufacturing method, crystal, crystal structure analysis method, crystallization screening method, crystallization screening apparatus
　本発明は、結晶製造方法、凍結結晶製造方法、結晶、結晶構造解析方法、結晶化スクリーニング方法、結晶化スクリーニング装置に関する。 The present invention relates to a crystal production method, a frozen crystal production method, a crystal, a crystal structure analysis method, a crystallization screening method, and a crystallization screening apparatus.
　タンパク質、核酸等の生体物質の立体構造を明らかにすることは、産業上極めて有用である。具体的には、例えば、前記生体物質の生体中における機能が明らかになるため、能率的な医薬品の開発が可能となる。 It is extremely useful industrially to clarify the three-dimensional structure of biological materials such as proteins and nucleic acids. Specifically, for example, since the function of the biological substance in the living body becomes clear, efficient drug development can be performed.
　タンパク質、核酸等の立体構造を解析するための方法としては、例えば結晶構造解析、特にＸ線回折像撮影による結晶構造解析が優れており、広く用いられている。この方法により解析するためには、検体（被解析物質）である前記タンパク質、核酸等の結晶を製造する必要がある。そのために、前記検体を含む溶液から前記検体の結晶を析出（結晶化）させる方法が行われている（特許文献１等）。
国際公開パンフレットＷＯ２００４／１０６５９８号公報 As a method for analyzing the three-dimensional structure of proteins, nucleic acids and the like, for example, crystal structure analysis, particularly crystal structure analysis by X-ray diffraction image photography is excellent and widely used. In order to perform analysis by this method, it is necessary to produce crystals of the protein, nucleic acid, etc., which are specimens (analytes to be analyzed). Therefore, a method of precipitating (crystallizing) crystals of the specimen from a solution containing the specimen (Patent Document 1, etc.) has been performed.
International Publication Pamphlet WO2004 / 106598
　前述のようにタンパク質、核酸等の溶液から結晶を析出（結晶化）させる結晶の製造方法には、非常に高度な技術が必要であり、困難を伴う。さらに、タンパク質、核酸等の結晶は、非常に脆く、結晶内部にひずみを生じやすいので、慎重に取り扱わなければ簡単に破壊されてしまうという問題がある。 As described above, a method for producing a crystal that precipitates (crystallizes) a crystal from a solution of protein, nucleic acid or the like requires a very advanced technique and is difficult. Furthermore, crystals of proteins, nucleic acids, and the like are very brittle and are likely to be distorted inside the crystals, so that there is a problem that they are easily destroyed unless handled carefully.
　そこで、本発明は、結晶を簡便に製造することが可能で、かつ、製造された結晶が取り扱いやすい結晶製造方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a method for producing a crystal, in which a crystal can be easily produced and the produced crystal is easy to handle.
　本発明者等は、前記課題を解決するために鋭意研究を重ねた。その結果、タンパク質、核酸等の生体物質の結晶製造方法において、前記生体物質の溶液に代えてゲル中から結晶を析出させることを見出した。前述の通り、タンパク質、核酸等の生体物質の結晶製造方法においては、従来、溶液から結晶を析出させることが一般に行われていた。これらの生体物質について、ゲル中から直接結晶を析出させる試みは、本発明者等がはじめて行った。 The inventors of the present invention have made extensive studies to solve the above problems. As a result, it has been found that in the method for producing crystals of biological materials such as proteins and nucleic acids, crystals are precipitated from the gel instead of the biological material solution. As described above, in the method for producing crystals of biological substances such as proteins and nucleic acids, conventionally, crystals are generally precipitated from a solution. The inventors of the present invention have made the first attempt to deposit crystals directly from the gel in these biological substances.
　すなわち、本発明の結晶製造方法は、
　生体物質の結晶を製造する方法であって、
　前記生体物質をゲル中で結晶化させる結晶化工程を含むことを特徴とする。 That is, the crystal production method of the present invention is:
A method for producing a crystal of biological material,
The method includes a crystallization step of crystallizing the biological material in a gel.
　さらに、本発明は、
　生体物質の結晶を凍結する凍結工程を含む凍結結晶の製造方法であって、
　前記本発明の結晶製造方法により、前記ゲルで被覆された前記被覆結晶を製造する被覆結晶製造工程をさらに含み、
　前記凍結工程において、前記被覆結晶を凍結することを特徴とする凍結結晶製造方法を提供する。 Furthermore, the present invention provides:
A method for producing a frozen crystal comprising a freezing step of freezing a crystal of biological material,
The method for producing a crystal according to the present invention further includes a coated crystal production process for producing the coated crystal coated with the gel,
In the freezing step, a frozen crystal production method is provided, wherein the coated crystal is frozen.
　さらに、本発明は、
　種結晶である生体物質結晶を前記生体物質の溶液中でさらに成長させる結晶成長工程を含む、前記生体物質の結晶の製造方法であって、
　前記結晶成長工程において、前記種結晶が、前記本発明の結晶製造方法により製造される、前記ゲルで被覆された被覆結晶であることを特徴とする結晶製造方法を提供する。なお、この結晶製造方法を、以下、「本発明の成長結晶製造方法」または単に「成長結晶製造方法」ということがある。 Furthermore, the present invention provides:
A method for producing a crystal of a biological material, comprising a crystal growth step of further growing a biological material crystal that is a seed crystal in a solution of the biological material,
In the crystal growth step, there is provided a crystal manufacturing method, wherein the seed crystal is a coated crystal coated with the gel manufactured by the crystal manufacturing method of the present invention. Hereinafter, this crystal production method may be referred to as “growth crystal production method of the present invention” or simply “growth crystal production method”.
　さらに、本発明は、前記本発明の結晶製造方法、前記本発明の凍結結晶製造方法、または前記本発明の成長結晶製造方法により製造される結晶を提供する。 Furthermore, the present invention provides a crystal produced by the crystal production method of the present invention, the frozen crystal production method of the present invention, or the growth crystal production method of the present invention.
　さらに、本発明は、
　生体物質の結晶を構造解析する方法であって、
　前記本発明の結晶製造方法により製造された被覆結晶または前記本発明の凍結結晶製造方法により製造された凍結結晶を構造解析する構造解析工程を含むことを特徴とする構造解析方法を提供する。 Furthermore, the present invention provides:
A method for structural analysis of a crystal of a biological material,
There is provided a structural analysis method comprising a structural analysis step of structural analysis of the coated crystal manufactured by the crystal manufacturing method of the present invention or the frozen crystal manufactured by the frozen crystal manufacturing method of the present invention.
　さらに、本発明は、
　生体物質の結晶化条件をスクリーニングする結晶化スクリーニング方法であって、
　前記生体物質の結晶を製造する結晶製造工程と、
　前記結晶製造工程における結晶化条件をスクリーニングするスクリーニング工程を含み、
　前記結晶製造工程において、本発明の結晶製造方法または本発明の凍結結晶製造方法により前記結晶を製造することを特徴とする結晶化スクリーニング方法を提供する。 Furthermore, the present invention provides:
A crystallization screening method for screening crystallization conditions of a biological material,
A crystal production process for producing crystals of the biological material;
Including a screening step of screening crystallization conditions in the crystal manufacturing step,
In the crystal production step, a crystallization screening method is provided, wherein the crystal is produced by the crystal production method of the present invention or the frozen crystal production method of the present invention.
　さらに、本発明は、
　生体物質の結晶化条件をスクリーニングする結晶化スクリーニング装置であって、
　前記生体物質の結晶を製造する結晶製造手段と、
　前記生体物質の結晶化条件をスクリーニングするスクリーニング手段とを含み、
　前記結晶製造手段が、ゲル中で前記生体物質の結晶を析出させる結晶化手段を含むことを特徴とする結晶化スクリーニング装置を提供する。 Furthermore, the present invention provides:
A crystallization screening apparatus for screening crystallization conditions of a biological material,
Crystal manufacturing means for manufacturing crystals of the biological material;
Screening means for screening the crystallization conditions of the biological material,
The crystallization screening apparatus is characterized in that the crystal production means includes a crystallization means for precipitating crystals of the biological material in a gel.
　本発明の結晶製造方法によれば、溶液中から結晶を析出させるよりも、前記生体物質の結晶を簡便に製造することが可能である。この理由は必ずしも明らかではないが、例えば、溶液中と異なり、生成した結晶がゲルにより固定されていることが原因と考えられる。より具体的には、例えば、生成した結晶がゲルに固定されているため、容器の壁等への衝突による破損、生成した結晶同士の衝突による多結晶化等が防止可能であり、格子欠陥の少ない単結晶に成長しやすいと考えられる。ただし、これらの考察は、可能な機構の一例を示すに過ぎず、本発明を何ら限定しない。 According to the crystal manufacturing method of the present invention, it is possible to easily manufacture a crystal of the biological substance, rather than precipitating the crystal from a solution. Although this reason is not necessarily clear, for example, unlike in the solution, it is considered that the generated crystal is fixed by a gel. More specifically, for example, since the generated crystal is fixed to the gel, it is possible to prevent damage due to collision with the wall of the container, polycrystallization due to collision between the generated crystals, and the like. It is thought that it is easy to grow into a few single crystals. However, these considerations are merely examples of possible mechanisms and do not limit the present invention in any way.
　また、本発明の凍結結晶製造方法によれば、前記結晶が凍結による損傷を受けにくい。さらに、本発明の構造解析方法によれば、前記ゲルで被覆された被覆結晶またはそれを凍結した前記凍結結晶を構造解析するため、結晶の取り扱いがしやすいという利点がある。 Moreover, according to the frozen crystal manufacturing method of the present invention, the crystal is not easily damaged by freezing. Furthermore, according to the structure analysis method of the present invention, since the structure analysis of the coated crystal coated with the gel or the frozen crystal frozen from the gel is performed, there is an advantage that the crystal is easy to handle.
　さらに、本発明の成長結晶製造方法によれば、前記ゲルで被覆された被覆結晶を種結晶として結晶を成長させるため、より大きな結晶を得ることができる。また、本発明の結晶は、前記本発明の結晶製造方法、前記本発明の凍結結晶製造方法、または前記本発明の成長結晶製造方法により製造されることで、例えば結晶構造解析等に向いた良好な特性を有する。ただし、本発明の結晶の製造方法は特に制限されず、同様の特性を有するのであれば他の任意の製造方法で製造された結晶でもよい。 Furthermore, according to the growth crystal manufacturing method of the present invention, since the crystal is grown using the coated crystal coated with the gel as a seed crystal, a larger crystal can be obtained. In addition, the crystal of the present invention is manufactured by the crystal manufacturing method of the present invention, the frozen crystal manufacturing method of the present invention, or the grown crystal manufacturing method of the present invention, so that it is suitable for crystal structure analysis, for example. It has special characteristics. However, the manufacturing method of the crystal of the present invention is not particularly limited, and may be a crystal manufactured by any other manufacturing method as long as it has similar characteristics.
　さらに、本発明の結晶化スクリーニング方法は、本発明の結晶製造方法により簡便に結晶を製造できることで、結晶製造条件をさほど厳密に設定しなくても良いため、スクリーニングをも簡便に行うことができる。さらに、本発明の結晶化スクリーニング装置は、同様の理由により、装置の構成を簡略化することが可能である。 Furthermore, since the crystallization screening method of the present invention can easily produce crystals by the crystal production method of the present invention, it is not necessary to set the crystal production conditions so strictly, and thus screening can be performed easily. . Furthermore, the crystallization screening apparatus of the present invention can simplify the configuration of the apparatus for the same reason.
図１Ａは、本発明の一実施例により得られた結晶の写真である。FIG. 1A is a photograph of a crystal obtained according to an embodiment of the present invention. 図１Ｂは、図１Ａの結晶を角度を変えて撮影した写真である。FIG. 1B is a photograph of the crystal of FIG. 1A taken at a different angle. 図２Ａは、図１の結晶のＸ線回折像を示す図である。FIG. 2A is a diagram showing an X-ray diffraction image of the crystal of FIG. 図２Ｂは、図２Ａの一部の拡大図である。FIG. 2B is an enlarged view of a part of FIG. 2A. 図３Ａは、本発明の他の一実施例により得られた結晶のＸ線回折像を示す図である。FIG. 3A is a diagram showing an X-ray diffraction image of a crystal obtained by another example of the present invention. 図３Ｂは、図３Ａの一部の拡大図である。FIG. 3B is an enlarged view of a part of FIG. 3A. 図４は、図３の結晶を別の条件により測定したときのＸ線回折像を示す図である。FIG. 4 is a diagram showing an X-ray diffraction image when the crystal of FIG. 3 is measured under different conditions. 図５Ａは、図４の結晶をさらに別の条件により測定したときのＸ線回折像を示す図である。FIG. 5A is a diagram showing an X-ray diffraction image when the crystal of FIG. 4 is measured under still another condition. 図５Ｂは、図５Ａの一部の拡大図である。FIG. 5B is an enlarged view of a part of FIG. 5A. 図６Ａは、図３～５の結晶を抗凍結剤に浸漬する直前の状態を示す写真である。FIG. 6A is a photograph showing a state immediately before the crystals of FIGS. 3 to 5 are immersed in the cryoprotectant. 図６Ｂは、図６Ａの結晶を抗凍結剤に浸漬してから５秒後の状態を示す写真である。FIG. 6B is a photograph showing a state after 5 seconds from immersing the crystal of FIG. 6A in the cryoprotectant. 図６Ｃは、図６Ａの結晶を抗凍結剤に浸漬してから１分後の状態を示す写真である。FIG. 6C is a photograph showing a state one minute after immersing the crystal of FIG. 6A in the cryoprotectant. 図６Ｄは、図６Ａの結晶を抗凍結剤に浸漬してから５分後の状態を示す写真である。FIG. 6D is a photograph showing a state after 5 minutes from immersing the crystal of FIG. 6A in the cryoprotectant. 図７Ａは、本発明のさらに別の一実施例により得られた結晶の写真である。FIG. 7A is a photograph of a crystal obtained by yet another example of the present invention. 図７Ｂは、図７Ａの結晶のＸ線回折像を示す図である。FIG. 7B is a diagram showing an X-ray diffraction image of the crystal of FIG. 7A. 図７Ｃは、図７Ｂの一部の拡大図である。FIG. 7C is an enlarged view of a part of FIG. 7B. 図８Ａは、本発明のさらに別の一実施例により得られた結晶の写真である。FIG. 8A is a photograph of a crystal obtained according to yet another example of the present invention. 図８Ｂは、図８Ａの結晶のＸ線回折像を示す図である。FIG. 8B is a diagram showing an X-ray diffraction image of the crystal of FIG. 8A. 図８Ｃは、図８Ｂの一部の拡大図である。FIG. 8C is an enlarged view of a part of FIG. 8B. 図９Ａは、比較例の結晶を抗凍結剤に浸漬する直前の状態を示す図である。FIG. 9A is a diagram showing a state immediately before the crystal of the comparative example is immersed in the cryoprotectant. 図９Ｂは、図９Ａの結晶を抗凍結剤に浸漬した直後の状態を示す図である。FIG. 9B is a diagram showing a state immediately after the crystal of FIG. 9A is immersed in an anti-freezing agent. 図１０Ａは、他の比較例の結晶を抗凍結剤に浸漬する直前の状態を示す図である。FIG. 10A is a diagram showing a state immediately before dipping a crystal of another comparative example in an antifreezing agent. 図１０Ｂは、図１０Ａの結晶を抗凍結剤に浸漬した直後の状態を示す図である。FIG. 10B is a diagram showing a state immediately after the crystal of FIG. 10A is immersed in an anti-freezing agent. 図１１は、本発明のさらに別の実施例および比較例の結晶の写真およびデータを示す図である。FIG. 11 is a diagram showing photographs and data of crystals of still another example and comparative example of the present invention. 図１２は、本発明のさらに別の実施例の結晶のＸ線回折像を示す図である。FIG. 12 is a diagram showing an X-ray diffraction image of a crystal according to still another example of the present invention. 図１３は、生体分子の結晶構造解析結果の一例を示す模式図である。FIG. 13 is a schematic diagram illustrating an example of a result of crystal structure analysis of a biomolecule. 図１４は、生体分子の結晶構造解析結果のさらに別の一例を示す模式図である。FIG. 14 is a schematic diagram showing still another example of the result of analyzing the crystal structure of a biomolecule. 図１５は、本発明の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 15 is a graph showing the crystallization screening result in one example of the present invention. 図１６は、本発明のさらに別の実施例における結晶化スクリーニング結果を示すグラフである。FIG. 16 is a graph showing crystallization screening results in still another example of the present invention. 図１７は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 17 is a graph showing crystallization screening results in still another example of the present invention. 図１８は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 18 is a graph showing crystallization screening results in still another example of the present invention. 図１９は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 19 is a graph showing crystallization screening results in still another example of the present invention. 図２０は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 20 is a graph showing crystallization screening results in still another example of the present invention. 図２１は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 21 is a graph showing a crystallization screening result in still another example of the present invention. 図２２は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示す写真およびグラフである。FIG. 22 is a photograph and a graph showing crystallization screening results in still another example of the present invention. 図２３は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示す写真およびグラフである。FIG. 23 is a photograph and a graph showing crystallization screening results in still another example of the present invention. 図２４は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示すグラフである。FIG. 24 is a graph showing crystallization screening results in still another example of the present invention. 図２５は、本発明のさらに別の一実施例における結晶化スクリーニング結果を示す写真である。FIG. 25 is a photograph showing a crystallization screening result in still another example of the present invention. 図２６は、本発明のさらに別の実施例における大腸菌由来タンパク質結晶を示す写真である。FIG. 26 is a photograph showing an E. coli-derived protein crystal in still another example of the present invention. 図２７は、本発明のさらに別の実施例におけるＤＮＡ結晶を示す写真である。FIG. 27 is a photograph showing a DNA crystal in still another example of the present invention. 図２８は、本発明のさらに別の実施例における、結晶化スクリーニング結果を示すグラフおよび結晶の写真である。FIG. 28 is a graph showing a crystallization screening result and a photograph of a crystal in still another example of the present invention. 図２９は、温度を低下させて被覆ゲルを除去した例を示す写真である。FIG. 29 is a photograph showing an example in which the coated gel is removed by lowering the temperature. 図３０は、実施例の結晶および比較例の結晶の乾燥耐性評価を示す写真である。FIG. 30 is a photograph showing the drying tolerance evaluation of the crystals of the example and the crystals of the comparative example. 図３１Ａは、さらに別の実施例および比較例の結晶の乾燥耐性評価を示す写真である。FIG. 31A is a photograph showing the drying tolerance evaluation of crystals of still another example and comparative example. 図３１Ｂは、図３１Ａの評価時間の経過後における、同じ実施例の結晶および比較例の結晶の乾燥耐性評価を示す写真である。FIG. 31B is a photograph showing the drying tolerance evaluation of the crystals of the same example and the comparative example after the elapse of the evaluation time of FIG. 31A. 図３２は、さらに別の実施例で製造した結晶において、周囲の溶液のゲル化前およびゲル化後の結晶状態を示す写真である。FIG. 32 is a photograph showing crystals before and after gelation of the surrounding solution in crystals produced in yet another example. 図３３は、実施例の結晶のＸ線回折像を示す図である。FIG. 33 is a diagram showing an X-ray diffraction image of the crystal of the example. 図３４は、さらに別の実施例の結晶のＸ線回折像を示す図である。FIG. 34 is a diagram showing an X-ray diffraction image of a crystal of still another example. 図３５は、ゲル被覆結晶のフェムト秒レーザーによる加工を示す写真である。FIG. 35 is a photograph showing processing of a gel-coated crystal with a femtosecond laser. 図３６は、フェムト秒レーザー加工したゲル被覆結晶の取り出しを示す写真である。FIG. 36 is a photograph showing removal of a gel-coated crystal that has been subjected to femtosecond laser processing. 図３７は、図３６の結晶の写真およびＸ線結晶構造解析データを示す図である。FIG. 37 shows a photograph of the crystal of FIG. 36 and X-ray crystal structure analysis data. 図３８は、成長結晶製造方法の一実施例を示す写真である。FIG. 38 is a photograph showing one example of a method for producing a grown crystal. 図３９は、成長結晶製造方法の別の一実施例を示す写真である。FIG. 39 is a photograph showing another example of the method for producing a grown crystal. 図４０は、遠心分離による濃度勾配を用いた結晶化スクリーニング方法の一例を模式的に示す図である。FIG. 40 is a diagram schematically illustrating an example of a crystallization screening method using a concentration gradient by centrifugation. 図４１は、図４０の方法による結晶化スクリーニング結果を示す写真である。FIG. 41 is a photograph showing a crystallization screening result by the method of FIG. 図４２は、結晶構造解析における結晶のマウント操作を例示する模式図である。FIG. 42 is a schematic view illustrating the crystal mounting operation in the crystal structure analysis. 図４３は、結晶構造解析における結晶のマウント操作に用いる機器を例示する模式図である。FIG. 43 is a schematic view illustrating an apparatus used for a crystal mounting operation in crystal structure analysis. 図４４は、本発明の結晶化スクリーニング装置の一例を示す模式図である。FIG. 44 is a schematic diagram showing an example of the crystallization screening apparatus of the present invention. 図４５は、本発明の結晶化スクリーニング装置（結晶化キット）のさらに別の一例を示す模式図である。FIG. 45 is a schematic diagram showing still another example of the crystallization screening apparatus (crystallization kit) of the present invention. 図４６は、本発明の結晶化スクリーニングの一例を示す模式図である。FIG. 46 is a schematic diagram showing an example of crystallization screening according to the present invention. 図４７は、アガロースゲルのゲル強度測定結果の一例を示すグラフである。FIG. 47 is a graph showing an example of a gel strength measurement result of an agarose gel. 図４８は、図４７のグラフのデータの一部を抜粋したグラフである。FIG. 48 is a graph obtained by extracting a part of the data of the graph of FIG.
　以下、本発明の実施形態について説明する。ただし、本発明は、以下の実施形態に限定されない。なお、本発明において、数値限定により発明を特定する場合、特に示さない限り、厳密にその数値でも良いし、約その数値でもよい。例えば、「０～９０℃」と記載した場合、特に示さない限り、厳密に０～９０℃でも良いし、約０℃～約９０℃でも良い。 Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments. In the present invention, when the invention is specified by numerical limitation, unless otherwise indicated, the numerical value may be strictly or approximately the numerical value. For example, when “0 to 90 ° C.” is described, it may be strictly 0 to 90 ° C. or about 0 ° C. to about 90 ° C. unless otherwise indicated.
［結晶製造方法］
　前述の通り、本発明の結晶製造方法は、生体物質を結晶化させる結晶化工程を含む、前記生体物質の結晶の製造方法であって、前記結晶化工程において、前記生体物質をゲル中で結晶化させることを特徴とする。 [Crystal production method]
As described above, the crystal production method of the present invention is a method for producing a crystal of a biological material, which includes a crystallization step of crystallizing the biological material, wherein the biological material is crystallized in a gel in the crystallization step. It is characterized by making it.
　本発明において、「生体物質」とは、生体由来の物質でも良いが、それと同一の構造を有する合成物質でも良く、または、生体由来物質と類似の構造を有する誘導体や人工物質であっても良いものとする。例えば、前記生体物質が生体高分子化合物の場合は、生体由来の高分子化合物でも、それと同一の構造を有する合成高分子化合物でも、または、生体由来高分子化合物と類似の構造を有する誘導体や人工高分子化合物であっても良い。前記生体物質がタンパク質の場合は、生体由来（天然由来）のタンパク質でも、合成タンパク質でも良いし、天然に存在する構造の天然タンパク質でも、天然に存在しない構造の人工タンパク質でもよい。前記生体物質がペプチドの場合は、生体由来（天然由来）のペプチドでも、合成ペプチドでも良いし、天然に存在する構造の天然ペプチドでも、天然に存在しない構造の人工ペプチドでもよい。前記生体物質が核酸の場合は、生体由来（天然由来）の核酸でも、合成核酸でも良いし、天然に存在する構造の天然核酸でも、天然に存在しない構造の人工核酸でもよい。前記生体物質が糖鎖の場合は、生体由来（天然由来）の糖鎖でも、合成糖鎖でも良いし、天然に存在する構造の天然糖鎖でも、天然に存在しない構造の人工糖鎖でもよい。前記生体物質は、特に制限されないが、例えば、生体高分子化合物、タンパク質、天然タンパク質、人工タンパク質、ペプチド、天然ペプチド、人工ペプチド、核酸、天然核酸、人工核酸、糖鎖、天然糖鎖、または人工糖鎖であることが好ましい。なお、天然核酸としては、特に制限されないが、例えば、ＤＮＡ、ＲＮＡ等があげられる。また、人工核酸としては、特に制限されないが、例えば、ＬＮＡ、ＰＮＡ等があげられる。また、本発明において、前記生体物質の分子量は特に制限されない。例えば、前記「生体高分子化合物」の分子量は、例えば、５０００以上であるが、これには限定されず、５０００未満であっても良い。また、前記生体物質がペプチドの場合、分子量は、例えば１０００以上であるが、これには限定されず、１０００未満であっても良い。 In the present invention, the “biological substance” may be a biological substance, but may be a synthetic substance having the same structure as it, or a derivative or an artificial substance having a structure similar to the biological substance. Shall. For example, when the biological substance is a biopolymer compound, it may be a polymer compound derived from a living body, a synthetic polymer compound having the same structure as it, or a derivative or artificial material having a structure similar to that of a bio-derived polymer compound. It may be a polymer compound. When the biological material is a protein, it may be a biologically derived (naturally-derived) protein, a synthetic protein, a naturally occurring natural protein, or a non-naturally occurring artificial protein. When the biological substance is a peptide, it may be a biologically derived (naturally-derived) peptide, a synthetic peptide, a naturally occurring peptide having a naturally occurring structure, or an artificial peptide having a non-naturally occurring structure. When the biological material is a nucleic acid, it may be a biologically derived (naturally-derived) nucleic acid, a synthetic nucleic acid, a naturally-occurring natural nucleic acid, or a non-naturally-occurring artificial nucleic acid. When the biological substance is a sugar chain, it may be a sugar chain derived from a living body (naturally derived), a synthetic sugar chain, a natural sugar chain having a naturally occurring structure, or an artificial sugar chain having a non-naturally occurring structure. . The biological material is not particularly limited, but for example, a biopolymer compound, protein, natural protein, artificial protein, peptide, natural peptide, artificial peptide, nucleic acid, natural nucleic acid, artificial nucleic acid, sugar chain, natural sugar chain, or artificial A sugar chain is preferred. The natural nucleic acid is not particularly limited, and examples thereof include DNA and RNA. The artificial nucleic acid is not particularly limited, and examples thereof include LNA and PNA. In the present invention, the molecular weight of the biological material is not particularly limited. For example, the molecular weight of the “biopolymer compound” is, for example, 5000 or more, but is not limited thereto, and may be less than 5000. Further, when the biological substance is a peptide, the molecular weight is, for example, 1000 or more, but is not limited thereto, and may be less than 1000.
　本発明の結晶製造方法は、前記結晶化工程に先立ち、前記生体物質の溶液を準備する溶液準備工程と、前記溶液をゲル化させて前記ゲルを調製するゲル化工程とをさらに含むことが好ましく、前記生体物質溶液がゲル化剤をさらに含むことがより好ましい。前記ゲル化剤は特に制限されないが、例えば、多糖類、増粘多糖類、タンパク質、および昇温時ゲル化型ゲルからなる群から選択される少なくとも一つであることが好ましく、アガロース、寒天、カラギーナン、ゼラチン、コラーゲン、ポリアクリルアミド、昇温時ゲル化型ポリアクリルアミドゲルからなる群から選択される少なくとも一つであることがより好ましい。ゲル化温度は特に制限されないが、結晶製造の行いやすさの観点から、例えば０～９０℃、好ましくは０～６０℃、より好ましくは０～３５℃である。 Preferably, the crystal production method of the present invention further includes a solution preparation step of preparing a solution of the biological material prior to the crystallization step, and a gelation step of preparing the gel by gelling the solution. More preferably, the biological material solution further contains a gelling agent. The gelling agent is not particularly limited, but is preferably at least one selected from the group consisting of polysaccharides, thickening polysaccharides, proteins, and gels at elevated temperature, agarose, agar, More preferably, it is at least one selected from the group consisting of carrageenan, gelatin, collagen, polyacrylamide, and gelled polyacrylamide gel at elevated temperature. The gelation temperature is not particularly limited, but is, for example, 0 to 90 ° C., preferably 0 to 60 ° C., more preferably 0 to 35 ° C. from the viewpoint of ease of crystal production.
　前記ゲル化剤は、例えば、低温でゲル化し高温でゾル化するゲルでも良いし、逆に低温でゾル化し高温でゲル化するゲルでも良い。低温でゾル化し高温でゲル化するゲルは、「昇温時ゲル化型ゲル」という。また、例えば、冷却により得られたゲルを再び昇温した時、あるいは昇温により得られたゲルを再び冷却した時、再びゾルに戻るゲルであることが好ましい。このようなゲル化剤は「熱可逆性ゲル」という。また、前記ゲル化剤は、例えば、ハイドロゲルでもオルガノゲルでも良いが、ハイドロゲルが好ましい。ハイドロゲルとしては、例えば、昇温時ゲル化型ハイドロゲルを用いることがより好ましい。昇温時ゲル化型ハイドロゲルは、低温でゲル化し高温でゾル化する一般的なゲルとは逆に、前述のように低温でゾル化し高温でゲル化する性質を持っている。このため、昇温時ゲル化型ハイドロゲルで被覆された被覆結晶は、例えば、乾燥に特に強い、冷却によりゲルを簡便に除去できる、などの利点を有する。昇温時ゲル化型ハイドロゲルとしては、特に制限されないが、例えば、メビオールジェルが挙げられる。メビオールジェルは、メビオール株式会社の昇温時ゲル化型ハイドロゲルの商品名であり、例えば下記の化学構造を有する。メビオールジェルは、昇温時ゲル化型ハイドロゲルとしての性質および熱可逆性ハイドロゲルとしての性質を有するポリアクリルアミドゲルである。
Figure JPOXMLDOC01-appb-C000001
 The gelling agent may be, for example, a gel that gels at a low temperature and forms a sol at a high temperature, or a gel that forms a sol at a low temperature and gels at a high temperature. Gels that sol at a low temperature and gel at a high temperature are referred to as “gels at elevated temperature”. Further, for example, a gel that returns to a sol again when the temperature of the gel obtained by cooling is increased again or when the temperature of the gel obtained by increasing temperature is cooled again is preferable. Such a gelling agent is called “thermo-reversible gel”. The gelling agent may be, for example, a hydrogel or an organogel, but is preferably a hydrogel. As the hydrogel, for example, it is more preferable to use a gelled hydrogel at elevated temperature. The gelled hydrogel at elevated temperature has the property of solling at a low temperature and gelling at a high temperature as described above, contrary to a general gel that gels at a low temperature and sols at a high temperature. For this reason, the coated crystal coated with the gelled hydrogel at elevated temperature has advantages such as being particularly strong in drying and capable of easily removing the gel by cooling. Although it does not restrict | limit especially as a gelatinization type hydrogel at the time of temperature rising, For example, a meviol gel is mentioned. Mebiol gel is a trade name of a gelled hydrogel produced by Mebiol Co., Ltd. and has the following chemical structure, for example. Meviol gel is a polyacrylamide gel having properties as a gelling hydrogel at elevated temperature and properties as a thermoreversible hydrogel.
Figure JPOXMLDOC01-appb-C000001
　本発明の結晶製造方法は、より具体的には、例えば、以下のようにして行うことができる。 More specifically, the crystal production method of the present invention can be performed, for example, as follows.
（１）溶液準備工程
　まず、前記生体物質を溶媒に溶かし、溶液とする。前記溶媒は特に制限されないが、例えば、公知の結晶製造方法に用いる溶媒と同じ物を用いてもよい。前記溶媒は、具体的には、例えば、水、エタノール、メタノール、アセトニトリル、アセトン、アニソール、イソプロパノール、酢酸エチル、酢酸ブチル、クロロホルム、シクロヘキサン、ジエチルアミン、ジメチルアセトアミド、ジメチルホルムアミド、トルエン、ブタノール、ブチルメチルエーテル、ヘキサン、ベンゼン、メチルエチルケトンからなる群から選択される少なくとも一つであり、好ましくは水である。前記生体物質の濃度は、特に制限されないが、例えば０．２～３００ｍｇ／ｍＬ、好ましくは０．５～１００ｍｇ／ｍＬ、より好ましくは１～５０ｍｇ／ｍＬである。また、必要に応じ、ｐＨ調整剤等を適宜加えてもよい。 (1) Solution preparation step First, the biological material is dissolved in a solvent to obtain a solution. Although the said solvent is not restrict | limited in particular, For example, you may use the same thing as the solvent used for the well-known crystal manufacturing method. Specific examples of the solvent include water, ethanol, methanol, acetonitrile, acetone, anisole, isopropanol, ethyl acetate, butyl acetate, chloroform, cyclohexane, diethylamine, dimethylacetamide, dimethylformamide, toluene, butanol, and butyl methyl ether. , Hexane, benzene, methyl ethyl ketone, at least one selected from the group consisting of methyl ethyl ketone, preferably water. The concentration of the biological substance is not particularly limited, but is, for example, 0.2 to 300 mg / mL, preferably 0.5 to 100 mg / mL, more preferably 1 to 50 mg / mL. Moreover, you may add a pH adjuster etc. suitably as needed.
（２）ゲル化工程
　次に、前記生体物質溶液にゲル化剤を加え、ゲル化させ、前記生体物質を含むゲルを調製する。ゲル化剤は、前記生体物質溶液中に直接加えても良いが、別途ゲル化剤溶液を調製した後、前記生体物質溶液に混合すると、均一に混合しやすいため好ましい。前記ゲル化剤溶液の溶媒は特に制限されないが、例えば、前記生体物質溶液と同じである。また、前記ゲル化剤溶液中におけるゲル化剤濃度は特に制限されないが、後述のゲル強度等の観点から、前記ゲル化剤溶液の全質量に対し、例えば０．６～５０質量％、好ましくは０．８～４０質量％、より好ましくは１．０～３０質量％、さらに好ましくは１．０～２５質量％、特に好ましくは１．０～２０質量％である。なお、ゲル化剤濃度と前記生体物質の結晶化のしやすさとの相関関係のメカニズムは不明であるが、例えば、後述する本発明の結晶化スクリーニング方法等を利用して、前記ゲル化剤濃度を適宜設定できる。前記生体物質溶液にゲル化剤を加えた後、ゲル化させる方法は特に制限されない。例えば、ゲル化温度よりも高い温度（例えば２０～４５℃）で前記ゲル化剤溶液を調製し、前記生体物質溶液と混合した後、ゲル化温度以下の温度で静置すれば良い。より具体的には、例えば、前記ゲル化剤溶液と前記生体物質溶液を混合した後、キャピラリーに封入し、キャピラリー中でゲル化させてもよい。これにより、ゲルがキャピラリーに封入された状態となる。また、熱可逆性ハイドロゲルの場合は、例えば前記とは逆に、低温で前記ゲル化剤溶液を調製し、前記生体物質溶液と混合した後、温度を上昇させてゲル化させてもよい。 (2) Gelation step Next, a gelling agent is added to the biological material solution to cause gelation, thereby preparing a gel containing the biological material. The gelling agent may be added directly to the biological material solution, but it is preferable to prepare a gelling agent solution separately and then mix it with the biological material solution because it is easy to mix uniformly. The solvent of the gelling agent solution is not particularly limited, but is the same as the biological material solution, for example. Further, the concentration of the gelling agent in the gelling agent solution is not particularly limited, but from the viewpoint of gel strength and the like described later, for example, 0.6 to 50% by mass, preferably The amount is 0.8 to 40% by mass, more preferably 1.0 to 30% by mass, still more preferably 1.0 to 25% by mass, and particularly preferably 1.0 to 20% by mass. The mechanism of the correlation between the gelling agent concentration and the ease of crystallization of the biological substance is unknown, but for example, the gelling agent concentration can be obtained by utilizing the crystallization screening method of the present invention described later. Can be set as appropriate. The method of gelling after adding a gelling agent to the biological material solution is not particularly limited. For example, the gelling agent solution may be prepared at a temperature higher than the gelation temperature (for example, 20 to 45 ° C.), mixed with the biological material solution, and then allowed to stand at a temperature equal to or lower than the gelation temperature. More specifically, for example, after the gelling agent solution and the biological material solution are mixed, they may be sealed in a capillary and gelled in the capillary. As a result, the gel is sealed in the capillary. In the case of a thermoreversible hydrogel, for example, contrary to the above, the gelling agent solution may be prepared at a low temperature, mixed with the biological material solution, and then gelled by increasing the temperature.
　前記ゲル化後のゲル強度は、後述する結晶化工程で析出した前記生体物質結晶を物理的衝撃から保護しやすい等の観点から、例えば１００Ｐａ以上、好ましくは２００Ｐａ以上、より好ましくは３００Ｐａ以上、さらに好ましくは５００Ｐａ以上、特に好ましくは１０００Ｐａ以上である。前記ゲル化剤のゲル強度は高いほど好ましく、上限は特に制限されないが、例えば、２０００００Ｐａ以下である。前記ゲル化後のゲル強度は、前記ゲル化剤濃度を調節することによって、適宜設定することができる。同じゲル化剤濃度における前記ゲル強度は、前記ゲル化剤の種類によって異なる。例えば、図４７のグラフに示すとおり、アガロースＩＩＩ、アガロースＳＰ（アガロースＳｅａ　Ｐｌａｑｕｅ）、アガロース９Ａ（いずれもタカラバイオ株式会社の商品名）は、同じ濃度であれば、ゲル化後は、通常、アガロースＩＩＩ＞アガロースＳＰ＞アガロース９Ａの順にゲル強度が高くなる。なお、図４８のグラフに、図４７におけるアガロース９Ａのみの測定結果を示す。図４７および４８において、横軸は前記各アガロースの濃度（質量％）であり、縦軸はゲル強度（ｇ／ｃｍ２）である。なお、１ｇ／ｃｍ２は、９８．０６６５Ｐａに相当する。また、ゲル化剤濃度が一定の臨界的濃度未満である場合、前記ゲル化剤を含む溶液が明確にゲル化しないため、ゲル強度は測定不能（グラフ上の記載としては０）である。しかしながら、ゲル化剤濃度が前記臨界的濃度以上になると、前記溶液が明確にゲル化することが可能であるため、ゲル強度が測定可能となる。本発明においては、前記ゲル化剤濃度を前記臨界的濃度以上にすることが好ましい。前記臨界的濃度は、アガロースＩＩＩ、アガロースＳＰ、アガロース９Ａの場合、図４７および図４８に示すとおり、いずれも、約０．６質量％である。 The gel strength after the gelation is, for example, 100 Pa or more, preferably 200 Pa or more, more preferably 300 Pa or more, from the viewpoint of easy protection of the biological material crystals deposited in the crystallization step described below from physical impact. Preferably it is 500 Pa or more, Most preferably, it is 1000 Pa or more. The gel strength of the gelling agent is preferably as high as possible, and the upper limit is not particularly limited, but is, for example, 200,000 Pa or less. The gel strength after gelation can be appropriately set by adjusting the gelling agent concentration. The gel strength at the same gelling agent concentration varies depending on the type of the gelling agent. For example, as shown in the graph of FIG. 47, agarose III, agarose SP (agarose sea plaque), and agarose 9A (all trade names of Takara Bio Inc.) are usually agarose after gelation at the same concentration. The gel strength increases in the order of III> agarose SP> agarose 9A. Note that the graph of FIG. 48 shows the measurement results of only agarose 9A in FIG. 47 and 48, the horizontal axis represents the concentration (mass%) of each agarose, and the vertical axis represents the gel strength (g / cm 2 ). Note that 1 g / cm 2 corresponds to 98.0665 Pa. Further, when the gelling agent concentration is less than a certain critical concentration, the gel strength cannot be measured (0 on the graph) because the solution containing the gelling agent does not clearly gel. However, when the gelling agent concentration is equal to or higher than the critical concentration, the gel strength can be measured because the solution can be clearly gelled. In the present invention, it is preferable that the concentration of the gelling agent is not less than the critical concentration. In the case of agarose III, agarose SP, and agarose 9A, the critical concentration is about 0.6% by mass as shown in FIG. 47 and FIG.
　なお、前記ゲル強度は、レオストレス　ＲＳ１　Ｒｈｅｏｍｅｔｅｒ（英弘精機株式会社のレオメーターの商品名）を動的粘弾性測定モードで用い、周波数１Ｈｚ、測定温度２０℃で測定した数値とする。図４７および図４８に示す測定値も、この方法で測定した。なお、ゲル溶解温度が２０℃よりも低いために２０℃でゲル状態にならない（ゾル状である）ゲルの場合は、前記ゲル強度は、前記ゲル溶解温度よりも低く５で割り切れる摂氏温度（例：１５℃、１０℃、５℃、０℃、－５℃）のうち最も高い測定可能温度での測定値とする。熱可逆性でありゲル溶解温度が２０℃よりも高いために２０℃でゲル状態にならない（ゾル状である）ゲルの場合は、前記ゲル強度は、前記ゲル溶解温度よりも高く５で割り切れる摂氏温度（例：２５℃、３０℃、３５℃）のうち最も低い測定可能温度での測定値とする。ただし、この測定方法は、前記ゲル強度の測定方法の一例であって、本発明は、この測定方法における工程および条件によって何ら限定されない。また、他のレオメーターを用いても、同じ測定モード、測定温度および周波数で測定すれば、誤差を除いて同じゲル強度測定値を得ることができる。 The gel strength is a numerical value measured at a frequency of 1 Hz and a measurement temperature of 20 ° C. using a rheostress RS1 Rheometer (trade name of Rheometer manufactured by Eihiro Seiki Co., Ltd.) in a dynamic viscoelasticity measurement mode. The measured values shown in FIGS. 47 and 48 were also measured by this method. In the case of a gel that is not in a gel state at 20 ° C. because the gel dissolution temperature is lower than 20 ° C. (in the form of a sol), the gel strength is lower than the gel dissolution temperature and can be divided by 5 (example: : 15 ° C, 10 ° C, 5 ° C, 0 ° C, -5 ° C). In the case of a gel that is thermoreversible and does not enter a gel state at 20 ° C. because the gel dissolution temperature is higher than 20 ° C. (the sol form), the gel strength is higher than the gel dissolution temperature and is divisible by 5 degrees Celsius. The measured value at the lowest measurable temperature among the temperatures (example: 25 ° C, 30 ° C, 35 ° C) is used. However, this measurement method is an example of the measurement method of the gel strength, and the present invention is not limited by the steps and conditions in this measurement method. Further, even if other rheometers are used, the same gel strength measurement value can be obtained except for errors if measurement is performed in the same measurement mode, measurement temperature and frequency.
（３）結晶化工程
　さらに、前記ゲル中から前記生体物質の結晶を析出させる。この方法は特に制限されない。例えば、前記ゲルを単に静置し、前記生体物質の結晶が析出するのを待ってもよい。また、例えば、前記ゲルを、沈殿化剤溶液に接触もしくは近接させた状態で静置してもよい。より具体的には、例えば、前記ゲルをキャピラリーに封入した後、前記沈殿化剤溶液に浸漬させてもよい。前記沈殿化剤は特に制限されないが、例えば、公知の結晶製造方法に用いる沈殿化剤と同じ物を用いてもよい。前記沈殿化剤は、例えば、塩化ナトリウム、塩化カルシウム、酢酸ナトリウム、酢酸アンモニウム、リン酸アンモニウム、硫酸アンモニウム、酒石酸カリウムナトリウム、クエン酸ナトリウム、PEG（ポリエチレングリコール）、塩化マグネシウム、カコジル酸ナトリウム、HEPES(2-〔4-(2-ヒドロキシエチル)-1-ピペラジニル〕エタンスルホン酸)、MPD(2-メチル-2,4-ペンタンジオール)、Tris-HCl（塩酸トリスヒドロキシメチルアミノメタン）からなる群から選択される少なくとも一つである。前記沈殿化剤溶液の溶媒は特に制限されないが、例えば、前記生体物質溶液と同じでもよい。前記沈殿化剤の濃度も特に制限されないが、例えば０．０００１～１０Ｍ、好ましくは０．０００５～８Ｍ、より好ましくは０．０００５～６Ｍである。また、前記沈殿化剤は、必要に応じ、ｐＨ調整剤等を適宜含んでいてもよい。なお、沈殿化剤は、「沈殿剤」とも言うことがある。また、上記においては、前記生体物質溶液を含むゲルを沈殿化剤溶液に接触もしくは近接させる方法を示したが、沈殿化剤を用いる結晶化方法は、これに限定されない。例えば、前記結晶化方法は、前記生体物質溶液にゲル化剤とともに沈殿化剤を混合しておき、ゲル化させて沈殿化剤を含むゲルとし、ゲル化後そのまま静置して結晶を析出させる方法でもよい。このような結晶化方法を「バッチ法」という。前記生体物質溶液の溶媒、前記生体物質の濃度、前記ゲル化剤の濃度、前記沈殿化剤の濃度等は、特に制限されないが、例えば、前記と同様である。 (3) Crystallization step Further, crystals of the biological material are precipitated from the gel. This method is not particularly limited. For example, the gel may be left still and wait for crystals of the biological material to precipitate. Further, for example, the gel may be allowed to stand in contact with or in close proximity to the precipitant solution. More specifically, for example, after the gel is sealed in a capillary, it may be immersed in the precipitant solution. The precipitating agent is not particularly limited, and for example, the same precipitating agent as used in a known crystal production method may be used. Examples of the precipitating agent include sodium chloride, calcium chloride, sodium acetate, ammonium acetate, ammonium phosphate, ammonium sulfate, potassium sodium tartrate, sodium citrate, PEG (polyethylene glycol), magnesium chloride, sodium cacodylate, HEPES (2 -[4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), MPD (2-methyl-2,4-pentanediol), Tris-HCl (trishydroxymethylaminomethane hydrochloride) Is at least one. The solvent for the precipitating agent solution is not particularly limited, and may be the same as the biological material solution, for example. The concentration of the precipitating agent is not particularly limited, but is, for example, 0.0001 to 10M, preferably 0.0005 to 8M, and more preferably 0.0005 to 6M. Moreover, the said precipitating agent may contain the pH adjuster etc. suitably as needed. The precipitating agent may also be referred to as “precipitating agent”. In the above description, the method of bringing the gel containing the biological material solution into contact with or in proximity to the precipitant solution is described. However, the crystallization method using the precipitant is not limited thereto. For example, in the crystallization method, the biological material solution is mixed with a precipitating agent together with a gelling agent, gelled to form a gel containing the precipitating agent, and left as it is after gelation to precipitate crystals. The method may be used. Such a crystallization method is called “batch method”. The solvent of the biological material solution, the concentration of the biological material, the concentration of the gelling agent, the concentration of the precipitating agent and the like are not particularly limited, but are the same as described above, for example.
　タンパク質等の生体物質の結晶化は、例えば、前記生体物質と沈殿化剤との混合比により制御することができる。この混合比をより詳細に制御することで、例えば、さらに高品質かつ大型の単結晶を得ることも可能である。本発明では、例えば、網目の分子構造を持ったゲル（例えばアガロースゲル）に前記のようにして沈殿化剤を拡散させることによって、濃度勾配を形成できる。これにより、前記生体物質溶液と沈殿化剤溶液を直接混合するよりも、前記混合比を容易に制御することが可能である。この場合、沈殿化剤の拡散速度が結晶成長速度に比べ十分遅いことがより好ましい。これにより、最適な混合比の条件で前記タンパク質等の生体物質の結晶化が実現できるため、結晶の製造がさらに簡便になる。また、これにより、本発明の製造方法は、様々な組み合わせの混合比を一気に検索することが可能な“コンビナトリアル結晶化技術”として、結晶化スクリーニング方法に応用することもできる。前記濃度勾配形成を利用したスクリーニングの具体的な方法およびそれに用いる装置は、例えば、後述の通りである。 Crystallization of biological materials such as proteins can be controlled by, for example, the mixing ratio of the biological material and the precipitating agent. By controlling the mixing ratio in more detail, for example, it is possible to obtain a higher quality and larger single crystal. In the present invention, for example, a concentration gradient can be formed by diffusing a precipitating agent as described above in a gel having a network molecular structure (for example, agarose gel). Thereby, it is possible to easily control the mixing ratio rather than directly mixing the biological material solution and the precipitant solution. In this case, it is more preferable that the diffusion rate of the precipitating agent is sufficiently slower than the crystal growth rate. Thereby, since the crystallization of the biological substance such as the protein can be realized under the optimum mixing ratio, the production of the crystal is further simplified. Accordingly, the production method of the present invention can also be applied to a crystallization screening method as a “combinatorial crystallization technique” capable of searching a mixture ratio of various combinations all at once. A specific screening method using the concentration gradient formation and an apparatus used therefor are as described below, for example.
　以上のようにして本発明の結晶製造方法を実施することができるが、本発明はこれに限定されない。本発明の結晶製造方法は、例えば、下記(i)または(ii)の方法により行っても良いし、それ以外の方法により行ってもよい。なお、本発明において、下記(i)を「カウンターディフュージョン法」と呼び、下記(ii)を「液体－固体法」と呼ぶものとする。
(i)前述のように、ゲル化温度よりも高い温度（例えば２０～４５℃）で前記ゲル化剤溶液を調製し、前記生体物質（タンパク質等）の溶液と混合し、キャピラリー中に封入してゲル化させる。その後、必要に応じ、沈殿化剤溶液と接触させる。
(ii)まず、ゲル（例えば、固形アガロース）を準備し、これに、別途準備した前記生体物質（タンパク質等）の溶液を浸透させ、ゲル中で前記生体物質を結晶化させる。必要に応じ、沈殿化剤を前記ゲルにさらに浸透させることにより、前記生体物質を結晶化させる。沈殿化剤は、溶液の状態でも、固形のまま用いてもよい。 Although the crystal manufacturing method of the present invention can be carried out as described above, the present invention is not limited to this. The crystal production method of the present invention may be performed, for example, by the following method (i) or (ii), or by other methods. In the present invention, the following (i) is referred to as “counter diffusion method”, and the following (ii) is referred to as “liquid-solid method”.
(i) As described above, the gelling agent solution is prepared at a temperature higher than the gelation temperature (for example, 20 to 45 ° C.), mixed with the solution of the biological material (protein etc.), and enclosed in a capillary. To gel. Thereafter, it is brought into contact with a precipitant solution as necessary.
(ii) First, a gel (for example, solid agarose) is prepared, and a solution of the biological material (protein, etc.) separately prepared is infiltrated into the gel to crystallize the biological material in the gel. If necessary, the biological material is crystallized by further infiltrating the gel with a precipitating agent. The precipitating agent may be used in the form of a solution or as a solid.
　さらに、本発明の結晶製造方法では、前述の通り、前記生体物質をゲル中で結晶化させる。この「ゲル中で結晶化させる」とは、例えば前述のように、ゲル化工程後にそのゲル中で前記生体物質を結晶化させてもよい。その他、前記「ゲル中で結晶化させる」とは、例えば、ゲル化剤を含む前記生体物質溶液が、ゲル化せずゾル状態の場合に、そのゾル状態のゲル化剤溶液中で前記生体物質を結晶化させることも含むものとする。メカニズムは明らかではないが、ゾル状態の溶液でも、ゲル化剤を含むことにより、結晶が析出しやすくなり、結晶の破損等が起こりにくくなるという本発明の効果が得られる場合があるためである。前記ゾル状態のゲル化剤溶液中で前記生体物質を結晶化させる方法は、例えば、前記ゲル化剤が熱可逆性ハイドロゲルである場合に特に有用である。また、例えば、前記生体物質および前記熱可逆性ハイドロゲルを含むゾル状態溶液を攪拌しながら前記生体物質を結晶化させ、その後前記溶液をゲル化させ、そのゲル中で前記生体物質結晶をさらに成長させることが特に好ましい。前記ゾル状態溶液の攪拌を行うことで、例えば、さらに前記生体物質結晶が成長しやすくなり、大きな結晶が得やすい等の利点がある。前記攪拌の速度は特に制限されないが、例えば１０～１０００ｒｐｍ、好ましくは３０～２００ｒｐｍ、より好ましくは５０～１００ｒｐｍである。前記攪拌の時間も特に制限されないが、例えば５分～６０日、好ましくは３０分～３０日、より好ましくは１時間～２０日である。前記攪拌時の前記ゾル状態溶液の温度も特に制限されないが、例えば０～４０℃、好ましくは２～３０℃、特に好ましくは４～２５℃である。 Furthermore, in the crystal manufacturing method of the present invention, as described above, the biological material is crystallized in a gel. This “crystallize in gel” may crystallize the biological material in the gel after the gelation step, for example, as described above. In addition, the term “crystallize in gel” means, for example, that the biological material solution containing a gelling agent is not gelled and is in a sol state, the biological material in the gelling agent solution in the sol state. And crystallizing. Although the mechanism is not clear, even if the solution is in a sol state, the inclusion of the gelling agent may lead to the effect of the present invention in that crystals are likely to precipitate and damage to the crystals is less likely to occur. . The method for crystallizing the biological material in the gelling agent solution in the sol state is particularly useful when, for example, the gelling agent is a thermoreversible hydrogel. In addition, for example, while stirring a sol solution containing the biological material and the thermoreversible hydrogel, the biological material is crystallized, and then the solution is gelled, and the biological material crystal is further grown in the gel. It is particularly preferred that By stirring the sol-state solution, for example, the biological material crystals are more likely to grow and large crystals can be easily obtained. The stirring speed is not particularly limited, but is, for example, 10 to 1000 rpm, preferably 30 to 200 rpm, more preferably 50 to 100 rpm. The stirring time is not particularly limited, but is, for example, 5 minutes to 60 days, preferably 30 minutes to 30 days, and more preferably 1 hour to 20 days. The temperature of the sol solution during the stirring is not particularly limited, but is, for example, 0 to 40 ° C., preferably 2 to 30 ° C., and particularly preferably 4 to 25 ° C.
　本発明の結晶製造方法により製造される結晶は、例えば、前記ゲルで被覆された被覆結晶であることが好ましい。この被覆結晶の製造条件は特に制限されないが、例えば、本発明の結晶製造方法において、前記ゲル中から前記生体物質の結晶を析出させることで、必然的に、前記ゲルで被覆された被覆結晶としてもよい。一般に、タンパク質等の生体物質の結晶は、脆く、かつ、乾燥等により変質しやすい。このため、生体物質結晶は、例えば、結晶構造解析の試料として供する操作（マウント）および種結晶として供する操作（Seeding）時など、慎重にかつ手早く操作を行わないと、物理的衝撃や乾燥により破損、変質等を起こしてしまうことが多い。これに対し、本発明における前記被覆結晶は、結晶がゲルで被覆されていることにより、乾燥や物理的衝撃に対する耐性が向上しており、結晶の変質、破損等が起こりにくい。このため、例えば、前記マウント操作やSeeding操作が極めて行いやすくなる。さらに、結晶の保存性も向上する。 The crystal produced by the crystal production method of the present invention is preferably, for example, a coated crystal coated with the gel. The production conditions of the coated crystal are not particularly limited. For example, in the crystal production method of the present invention, the crystal of the biological material is precipitated from the gel, so that the coated crystal covered with the gel is necessarily formed. Also good. In general, crystals of biological materials such as proteins are brittle and are easily altered by drying or the like. For this reason, biological material crystals are damaged by physical impact or drying unless they are operated carefully and quickly, for example, when they are used as samples for crystal structure analysis (mounting) and when they are used as seed crystals (seeding). In many cases, it will cause alteration. On the other hand, the coated crystal in the present invention is improved in resistance to drying and physical impact because the crystal is coated with a gel, so that the crystal is hardly deteriorated or damaged. For this reason, for example, the mounting operation and the seeding operation are extremely easy to perform. Furthermore, the preservability of crystals is improved.
　なお、前記被覆結晶において、結晶を被覆している前記ゲルは、例えば、後述の結晶構造解析において測定ノイズの原因となるなどの支障がある場合は、あらかじめ除去することが好ましい。除去方法は特に制限されない。例えば、前記ゲルが熱可逆性ハイドロゲルであれば、前述の通り、冷却により前記ゲルがゾル化するため、簡便に除去できる。ゾル化させるための冷却温度は特に制限されないが、メビオールジェルの場合、例えば、１５℃以下である。また、前記被覆結晶を適宜な方法で加工し、前記ゲルを含まない結晶だけを取り出すこともできる。前記加工の方法は特に制限されないが、例えば、レーザー光を用いた加工がある。前記レーザー光も特に制限されないが、フェムト秒レーザーを用いることが特に好ましい。フェムト秒レーザーは、集光点付近のみで加工を行うことが可能であるため、例えば、結晶製造（育成）用容器を密封したままで簡便に加工できる等の利点があるためである。また、レーザー光を用いた加工によれば、例えば、結晶を、構造解析等の用途に適した大きさや形状に切り出すこともできる。このような加工工程により、結晶を適切な大きさや形状に切り出すことで、前記適切な大きさや形状の結晶を製造することもできる。また、例えば、前記加工工程により、前記被覆結晶からゲルのみを適切に除去し、結晶部分のみを残した加工結晶を製造することもできる。すなわち、前記加工結晶は、ゲルで被覆された被覆結晶でも良いし、ゲルで被覆されていなくても良い。 In the coated crystal, the gel covering the crystal is preferably removed in advance if there is a problem such as causing measurement noise in the crystal structure analysis described later. The removal method is not particularly limited. For example, if the gel is a thermoreversible hydrogel, as described above, the gel is made into a sol by cooling and can be easily removed. The cooling temperature for making the sol is not particularly limited, but in the case of meviol gel, for example, it is 15 ° C. or lower. Further, the coated crystal can be processed by an appropriate method, and only the crystal not containing the gel can be taken out. Although the processing method is not particularly limited, for example, there is processing using laser light. The laser light is not particularly limited, but it is particularly preferable to use a femtosecond laser. This is because the femtosecond laser can be processed only in the vicinity of the condensing point, and thus has an advantage that it can be easily processed while the crystal manufacturing (growing) container is sealed, for example. In addition, according to processing using laser light, for example, a crystal can be cut into a size and shape suitable for applications such as structural analysis. The crystal having the appropriate size and shape can be manufactured by cutting the crystal into an appropriate size and shape by such a processing step. In addition, for example, by the processing step, it is possible to appropriately remove only the gel from the coated crystal and manufacture a processed crystal that leaves only the crystal portion. That is, the processed crystal may be a coated crystal coated with a gel or may not be coated with a gel.
　なお、レーザー光によるこのような加工は、ゲルで被覆されていない結晶に対しても行うことができる。しかしながら、前記被覆結晶であれば、結晶がゲルにより固定および保護されているため、結晶が加工しやすく損傷を受けにくい、加工の際のデブリ（切りくずや結晶の破片など）が拡散せず結晶表面に再付着しにくい、などの利点がある。 It should be noted that such processing with laser light can also be performed on crystals that are not coated with gel. However, in the case of the coated crystal, since the crystal is fixed and protected by the gel, the crystal is easy to process and is not easily damaged, and debris (such as chips and crystal fragments) during processing does not diffuse. There are advantages such as being difficult to reattach to the surface.
　本発明の結晶製造方法によれば、例えば、従来よりも高い確率で高品質かつ大型の結晶が得られ、かつ、前述の濃度勾配形成を利用したスクリーニング法（濃度勾配法）によれば、結晶化条件の広範な探索（コンビナトリアル探索）が可能である。これらに関しては、例えば、従来、タンパク質等の結晶化で最も良く用いられてきた蒸気拡散法よりも優れた結果を得ることもできる。 According to the crystal manufacturing method of the present invention, for example, a high-quality and large-sized crystal can be obtained with a higher probability than in the past, and according to the screening method (concentration gradient method) using the above-described concentration gradient formation, A wide range of search conditions (combinatorial search) is possible. With respect to these, for example, it is possible to obtain results superior to the vapor diffusion method that has been conventionally most commonly used for crystallization of proteins and the like.
　また、本発明の結晶製造方法によれば、例えば、前記ゲル中から前記生体物質の結晶を析出させることで、以下のような利点が得られる。 Also, according to the crystal manufacturing method of the present invention, for example, the following advantages can be obtained by precipitating crystals of the biological material from the gel.
　構造ゲノム科学の分野におけるタンパク質結晶の構造解析等では、結晶のマウントに高度な技能を有する。例えば、低温における結晶構造解析を行うに際しては、抗凍結条件下に置いた結晶をナイロン糸を用いたループ状のマウント器具を用いて溶液ごとすくい取り、表面張力でループ内に結晶を保持した状態で凍結および測定を行う方法が従来から行われている。この方法では結晶の周りを抗凍結剤溶液が取り囲むので、測定中に結晶を非接触で保持が可能である。その反面、結晶の取り出し時にループの接触等による物理的な損傷を与える可能性がある。すなわち、溶液中で得られた結晶は、結晶マウントの際、溶液中を自由に移動するため、前記ループ等への間接・直接的な接触により、Ｘ線測定前に損傷を受けるおそれがある。このため、精度良い測定を行うにはループの取り扱いについての熟練した技能が要求される。 In the field of structural genomics, protein crystal structure analysis has advanced skills in crystal mounting. For example, when conducting crystal structure analysis at low temperatures, the crystals placed under anti-freezing conditions are scooped together with the solution using a loop-shaped mounting device using nylon thread, and the crystals are held in the loop by surface tension. Conventionally, a method of freezing and measuring in the above has been performed. In this method, since the cryoprotectant solution surrounds the crystal, the crystal can be held in a non-contact manner during the measurement. On the other hand, there is a possibility of physical damage due to contact of the loop when the crystal is taken out. That is, since the crystal obtained in the solution freely moves in the solution when the crystal is mounted, it may be damaged before the X-ray measurement by indirect or direct contact with the loop or the like. For this reason, in order to perform measurement with high accuracy, a skilled skill in handling the loop is required.
　一方、本発明の結晶製造方法では、ゲル中から前記生体物質（タンパク質等）の結晶を析出させる。このため、前記生体物質結晶がゲルで固定された状態となる。したがって、前記生体物質結晶がゲルで固定され移動が阻害されること、ゲルで保護され損傷を受けにくいこと等により、マウント操作が容易であり、再現性の高いマウント操作が可能となる。これによれば、例えば、結晶マウント工程を自動化することにより、従来達成不可能であったタンパク質結晶等のＸ線構造解析のフルオートメーション化も達成可能となる。また、固相（ゲル中）に前記生体物質（タンパク質等）の結晶を析出させることで、得られた結晶はゲルに被覆されるため、結晶の凍結と簡便なマウント操作が可能となり、結晶の質を低下させずに凍結できるため高精度データが得られる。 On the other hand, in the crystal production method of the present invention, crystals of the biological substance (proteins, etc.) are precipitated from the gel. For this reason, the said biological material crystal will be in the state fixed with the gel. Therefore, the mounting operation is easy and the mounting operation is highly reproducible due to the fact that the biological material crystal is fixed by the gel and the movement is inhibited, and the biological material crystal is protected by the gel and is not easily damaged. According to this, for example, by automating the crystal mounting process, it is possible to achieve full automation of X-ray structural analysis of protein crystals and the like, which could not be achieved conventionally. In addition, by precipitating crystals of the biological material (proteins, etc.) on the solid phase (in the gel), the obtained crystals are covered with the gel, so that the crystals can be frozen and a simple mounting operation can be performed. High accuracy data can be obtained because it can be frozen without degrading quality.
［凍結結晶製造方法］
　次に、本発明の凍結結晶製造方法は、前述の通り、生体物質の結晶を凍結する凍結工程を含む凍結結晶の製造方法であって、前記本発明の結晶製造方法により、前記ゲルで被覆された前記被覆結晶を製造する被覆結晶製造工程をさらに含み、前記凍結工程において、前記被覆結晶を凍結することを特徴とする。これ以外には、本発明の凍結結晶製造方法は特に制限されないが、前記凍結工程に先立ち、前記被覆結晶を抗凍結剤に浸漬させる浸漬工程をさらに含むことが好ましい。 [Frozen crystal production method]
Next, the frozen crystal manufacturing method of the present invention is a frozen crystal manufacturing method including a freezing step of freezing the crystal of biological material, as described above, and is coated with the gel by the crystal manufacturing method of the present invention. The method further comprises a coated crystal manufacturing step for manufacturing the coated crystal, wherein the coated crystal is frozen in the freezing step. Other than this, the method for producing frozen crystals of the present invention is not particularly limited, but it is preferable that the method further includes an immersion step of immersing the coated crystals in an antifreezing agent prior to the freezing step.
　例えば、タンパク質のＸ線構造解析を行う場合には、結晶を凍結した状態（例えば100K以下）でＸ線回折データを収集することが、従来から行われている。その理由としては、例えば、タンパク質結晶を凍結した状態で測定することで、放射線（例えば、高分解能データを得るためのシンクロトロン放射光）による前記結晶の損傷を劇的に抑制するという理由が挙げられる。また、例えば、サイズが極めて小さい結晶は、キャピラリーに封入して測定することが難しいため、キャピラリーへの封入に代えて前記凍結により、放射線による前記結晶の損傷を抑制する。しかし、この場合、タンパク質結晶の周りの水分子が氷へと変化し、結晶自体を損傷させてしまうため、良好なＸ線回折像を得ることができず、回折データの質や分解能を大きく低下させる場合がある。この機構は必ずしも明らかではないが、例えば、食品を冷凍した際に風味や品質が劣化する場合があるのと同様、凍結により結晶が変質してしまうためと考えられる。そのため、抗凍結剤と呼ばれる試薬を適量、結晶化母液に加え、その中にタンパク質結晶を浸す操作を行う事により、水をアモルファス状で凍結させる事が行われている。しかし、タンパク質、核酸等の結晶は環境の変化に弱いため、前記抗凍結剤の添加で結晶化母液に変化が生じることによって、致命的な損傷を受ける場合がある。そのため、前記抗凍結剤の添加により、かえって良好なＸ線回折点が得られず、構造解析に至らない場合がある。 For example, when performing X-ray structural analysis of a protein, it has been conventionally performed to collect X-ray diffraction data in a frozen state (for example, 100K or less). The reason is, for example, that the damage to the crystal due to radiation (for example, synchrotron radiation for obtaining high resolution data) is dramatically suppressed by measuring the protein crystal in a frozen state. It is done. In addition, for example, since a crystal having a very small size is difficult to be measured by being enclosed in a capillary, the freezing instead of the encapsulation in the capillary suppresses damage to the crystal due to radiation. However, in this case, water molecules around the protein crystal change to ice and damage the crystal itself, so that a good X-ray diffraction image cannot be obtained, and the quality and resolution of diffraction data are greatly reduced. May be allowed Although this mechanism is not necessarily clear, for example, it is considered that the crystals are altered by freezing as in the case where the flavor and quality are deteriorated when the food is frozen. Therefore, an appropriate amount of a reagent called an anti-freezing agent is added to the crystallization mother liquor, and the operation of immersing the protein crystal in it is performed to freeze water in an amorphous state. However, since crystals of proteins, nucleic acids and the like are vulnerable to environmental changes, there may be fatal damage due to changes in the crystallization mother liquor caused by the addition of the cryoprotectant. For this reason, the addition of the anti-freezing agent may result in failure to obtain a good X-ray diffraction point, which may not lead to structural analysis.
　本発明の凍結結晶製造方法により得られた凍結結晶は、例えば、凍結で生じた氷による損傷が抑制される。この理由は必ずしも明らかでないが、前記被覆結晶および凍結結晶がゲルで保護されているためと考えられる。また、例えば、前記ゲルにより、Ｘ線等による損傷も抑制されると考えられる。また、前記被覆結晶を抗凍結剤に浸漬させる浸漬工程をさらに含むことで、凍結による結晶損傷がさらに抑制される。さらに、前記被覆結晶がゲルで被覆されていることにより、抗凍結剤による結晶の損傷を大幅に抑制することができる。 In the frozen crystal obtained by the frozen crystal production method of the present invention, for example, damage caused by ice caused by freezing is suppressed. Although this reason is not necessarily clear, it is considered that the coated crystal and the frozen crystal are protected with a gel. In addition, for example, the gel is considered to suppress damage caused by X-rays or the like. Moreover, the crystal | crystallization damage by freezing is further suppressed by further including the immersion process which immerses the said covering crystal | crystallization in an antifreezing agent. Further, since the coated crystal is coated with a gel, damage to the crystal due to the cryoprotectant can be significantly suppressed.
　本発明の凍結結晶製造方法において、抗凍結剤は、特に制限されないが、有機化合物が好ましく、例えば、グリセロール（グリセリン）、MPD(2-Methyl-2,4-PentaneDiol)、DMSO（ジメチルスルホキシド）、PEG（ポリエチレングリコール）、および酢酸リチウムからなる群から選択される少なくとも一つである。これらは、そのまま用いても良いし、水溶液としてもよい。従来の凍結結晶製造方法では、前述の通り、抗凍結剤による結晶の損傷が著しいため、例えば、結晶化母液中に加える抗凍結剤の量（濃度）を微調整し、最適な条件をスクリーニングすることが必要であった。また、結晶化の対象となる生体物質（タンパク質等）の種類に応じて、最適な抗凍結剤の探索も必要であった。本発明において、前記抗凍結剤の濃度は特に制限されない。前述の通り、本発明によれば抗凍結剤による結晶の損傷を大幅に抑制することができるので、例えば、１００％のDMSO、PEG、あるいはそれらの高濃度水溶液（例えば５０～６０質量％）に前記生体物質の結晶を直接浸漬することもできる。本発明において、抗凍結剤の種類自体は、例えば、従来と同じものを用いてもよい。 In the method for producing frozen crystals of the present invention, the cryoprotectant is not particularly limited, but is preferably an organic compound, such as glycerol (glycerin), MPD (2-Methyl-2,4-PentaneDiol), DMSO (dimethyl sulfoxide), It is at least one selected from the group consisting of PEG (polyethylene glycol) and lithium acetate. These may be used as they are or as an aqueous solution. In the conventional method for producing frozen crystals, as described above, since the damage of crystals due to the cryoprotectant is significant, for example, the amount (concentration) of the cryoprotectant added to the crystallization mother liquor is finely adjusted, and optimal conditions are screened. It was necessary. In addition, it is necessary to search for an optimal cryoprotectant according to the type of biological material (protein, etc.) to be crystallized. In the present invention, the concentration of the cryoprotectant is not particularly limited. As described above, according to the present invention, the crystal damage caused by the cryoprotectant can be greatly suppressed. For example, 100% DMSO, PEG, or a high concentration aqueous solution thereof (for example, 50 to 60% by mass) can be used. Crystals of the biological material can also be directly immersed. In the present invention, the type of anti-freezing agent itself may be, for example, the same as that conventionally used.
　なお、従来の生体物質結晶の構造解析において、例えば、図１３の模式図に示すように、抗凍結剤分子が前記生体物質の活性部位に結合し、結晶中に残留してしまう場合があった。同図は、グルコースイソメラーゼ（ＧＩ）のX線結晶構造解析において、抗凍結剤としてグリセロールを用い、分解能０．９４Åで測定した場合の構造解析結果の一例を示す模式図である。図中の「２ＧＬＫ」はＰＤＢ　ＩＤ（プロテインデータバンクの登録番号）であり、グルコースイソメラーゼ（ＧＩ）を意味する。このように、結晶中に抗凍結剤分子が残留すると、活性部位を塞ぎ、例えば、薬物設計等の際に不利となる。一方、本発明の被覆結晶を用いれば、例えば、図１４の模式図に示すように、活性部位に抗凍結剤が結合せずフリーな状態の構造情報が得られ、薬物設計等に有利である。前述の通り、従来の凍結結晶製造方法では、抗凍結剤による結晶の損傷が著しいため、結晶の損傷を起こしにくい抗凍結剤の種類は、ごく限られていた。このため、結晶の損傷を起こしにくい抗凍結剤の中から、結晶中に残留しにくい抗凍結剤を選択することも困難であった。しかしながら、本発明の凍結結晶製造方法によれば、抗凍結剤による結晶の損傷が抑制されているため、選択できる抗凍結剤の幅が広く、結晶中に残留しにくい抗凍結剤を適切に選択することもできるのである。例えば、グルコースイソメラーゼに対しては、必ずしも明らかではないが、グリセロールよりも酢酸リチウムの方が結晶中に残留しにくいと考えられる。ただし、この説明はあくまでも例示であって、本発明を何ら限定せず、例えば、本発明に用いる抗凍結剤としてグリセロールが不適切であるということを意味しない。 In the conventional structural analysis of a biological material crystal, for example, as shown in the schematic diagram of FIG. 13, the cryoprotectant molecule may bind to the active site of the biological material and remain in the crystal. . This figure is a schematic diagram showing an example of a structural analysis result when glycerol is used as an anti-freezing agent and measurement is performed at a resolution of 0.94 mm in X-ray crystal structure analysis of glucose isomerase (GI). “2GLK” in the figure is a PDB ID (protein data bank registration number), which means glucose isomerase (GI). Thus, if the cryoprotectant molecules remain in the crystal, the active site is blocked, which is disadvantageous for drug design, for example. On the other hand, when the coated crystal of the present invention is used, for example, as shown in the schematic diagram of FIG. 14, the freezing structure is not bonded to the active site and free structure information is obtained, which is advantageous for drug design and the like. . As described above, in the conventional frozen crystal manufacturing method, the crystal damage due to the cryoprotectant is significant, and therefore the types of antifreeze agents that are less likely to cause crystal damage have been limited. For this reason, it has been difficult to select an anti-freezing agent that hardly remains in the crystal from among the anti-freezing agents that hardly cause crystal damage. However, according to the method for producing a frozen crystal of the present invention, the damage of the crystal caused by the cryoprotectant is suppressed, so that the range of the cryoprotectant that can be selected is wide, and the cryoprotectant that does not easily remain in the crystal is appropriately selected. You can also do it. For example, for glucose isomerase, although not necessarily clear, it is considered that lithium acetate is less likely to remain in the crystal than glycerol. However, this explanation is merely an example, and does not limit the present invention at all. For example, it does not mean that glycerol is inappropriate as an antifreezing agent used in the present invention.
［成長結晶製造方法］
　本発明の成長結晶製造方法は、前述の通り、種結晶である生体物質結晶を前記生体物質の溶液中でさらに成長させる結晶成長工程を含み、前記種結晶が、前記ゲルで被覆された被覆結晶である。これ以外には、本発明の成長結晶製造方法は特に制限されない。例えば、前記結晶成長工程における前記生体物質溶液の濃度、温度等の各種条件は、従来技術において種結晶を用いて結晶を成長させる方法等を参考にして適宜設定してもよい。前記生体物質溶液の好ましい濃度は、前記生体物質の種類等により異なるが、成長の結晶しやすさの観点からは、なるべく高濃度であることが好ましい。温度も特に制限されず、例えば、室温（約５～３５℃程度）で静置しても良いし、冷蔵庫等で５℃以下、あるいは０℃以下の低温に冷却しても良い。前記結晶成長工程の時間も特に制限されず、望ましい結晶が析出するまで適宜静置しておけば良い。 [Growth crystal production method]
As described above, the growth crystal manufacturing method of the present invention includes a crystal growth step of further growing a biological material crystal as a seed crystal in a solution of the biological material, and the seed crystal is coated with the gel. It is. Other than this, the growth crystal production method of the present invention is not particularly limited. For example, various conditions such as the concentration and temperature of the biological material solution in the crystal growth step may be appropriately set with reference to a method of growing a crystal using a seed crystal in the prior art. The preferable concentration of the biological material solution varies depending on the type of the biological material, but is preferably as high as possible from the viewpoint of ease of crystallization of growth. The temperature is not particularly limited, and may be left at room temperature (about 5 to 35 ° C.), or may be cooled to a low temperature of 5 ° C. or lower or 0 ° C. or lower with a refrigerator or the like. The time for the crystal growth step is not particularly limited, and may be allowed to stand as appropriate until a desired crystal is precipitated.
　種結晶を溶液中でさらに成長させる結晶製造方法は広く行われているが、タンパク質等の生体物質の場合、結晶が脆いために、種結晶を前記生体物質溶液中に移動させる際、物理的衝撃による損傷等が起こるおそれがあった。また、前記種結晶を前記生体物質溶液中に移動させた後、前記種結晶の周りの溶液の濃度や浸透圧の変化により、前記種結晶が溶解してしまい、結晶成長が起こらないおそれがあった。しかし、本発明の成長結晶製造方法によれば、前記種結晶が、前記ゲルで被覆された被覆結晶であるため、物理的衝撃による損傷のおそれが激減する。また、前記種結晶が、前記ゲルで被覆された被覆結晶であるために、前記種結晶の溶解も抑制され、結晶が成長しやすい。これにより、本発明の成長結晶製造方法によれば、例えば中性子線結晶構造解析等の各種用途に向いた大型の結晶を、簡便な操作により、高い確実性で得ることができる。 A crystal manufacturing method for further growing a seed crystal in a solution is widely performed. However, in the case of a biological material such as a protein, since the crystal is brittle, a physical impact is caused when the seed crystal is moved into the biological material solution. There was a risk of damage. In addition, after the seed crystal is moved into the biological material solution, the seed crystal may be dissolved due to a change in the concentration or osmotic pressure of the solution around the seed crystal, and crystal growth may not occur. It was. However, according to the growth crystal manufacturing method of the present invention, since the seed crystal is a coated crystal coated with the gel, the risk of damage due to physical impact is drastically reduced. Further, since the seed crystal is a coated crystal coated with the gel, dissolution of the seed crystal is also suppressed, and the crystal is likely to grow. Thereby, according to the growth crystal manufacturing method of the present invention, a large crystal suitable for various applications such as neutron beam crystal structure analysis can be obtained with high reliability by a simple operation.
［構造解析方法］
　次に、本発明の構造解析方法は、前述の通り、生体物質の結晶を構造解析する方法であって、本発明の結晶製造方法により製造された被覆結晶または本発明の凍結結晶製造方法により製造された凍結結晶を構造解析する構造解析工程を含むことを特徴とする。前記構造解析工程において、前記結晶を構造解析する方法は特に制限されないが、Ｘ線結晶構造解析または中性子線結晶構造解析によることが好ましい。本発明の結晶製造方法によれば、前述の通り、結晶の破損、多結晶化等が防止できるため、中性子線結晶構造解析に適した大きい結晶を得ることができる。 [Structural analysis method]
Next, as described above, the structural analysis method of the present invention is a method for structural analysis of a crystal of a biological material, which is manufactured by a coated crystal manufactured by the crystal manufacturing method of the present invention or a frozen crystal manufacturing method of the present invention. And a structural analysis step for structural analysis of the frozen crystals. In the structural analysis step, a method for analyzing the structure of the crystal is not particularly limited, but it is preferable to use X-ray crystal structure analysis or neutron beam crystal structure analysis. According to the crystal manufacturing method of the present invention, as described above, damage to the crystal, crystallization, and the like can be prevented, and thus a large crystal suitable for neutron beam crystal structure analysis can be obtained.
　上記以外には、本発明の構造解析方法は特に制限されず、従来の結晶構造解析方法、Ｘ線結晶構造解析方法または中性子線結晶構造解析方法と同様に行ってもよい。本発明の構造解析方法において構造解析する生体物質（タンパク質等）の結晶は、本発明の結晶製造方法により製造された被覆結晶または本発明の凍結結晶製造方法により製造された凍結結晶であることにより、高品質かつ大型の結晶とすることができる。これにより、本発明の構造解析方法においては、高精度のデータ（例えば、Ｘ線回折データ）を再現性良く得ることができる。その他に、本発明の結晶製造方法および凍結結晶製造方法の項で述べたような利点もある。 Other than the above, the structure analysis method of the present invention is not particularly limited, and may be performed in the same manner as the conventional crystal structure analysis method, X-ray crystal structure analysis method, or neutron beam crystal structure analysis method. Crystals of biological substances (proteins etc.) subjected to structural analysis in the structural analysis method of the present invention are coated crystals manufactured by the crystal manufacturing method of the present invention or frozen crystals manufactured by the frozen crystal manufacturing method of the present invention. High quality and large crystals. Thereby, in the structure analysis method of the present invention, highly accurate data (for example, X-ray diffraction data) can be obtained with good reproducibility. In addition, there are advantages as described in the section of the crystal production method and the frozen crystal production method of the present invention.
　本発明の構造解析方法は、前述の通り特に制限されず、どのような方法でもよいが、例えば以下のようにして行うことができる。図４２の上部に第一のスキームを、下部に第二のスキームを、それぞれ別個に示す。第一のスキームは本発明の構造解析方法の第一の例を示し、第二のスキームは本発明の構造解析方法の第二の例を示す。同図中の文言は、説明の便宜のための例示であって、本発明を何ら限定しない。 The structure analysis method of the present invention is not particularly limited as described above, and any method may be used. For example, the structure analysis method can be performed as follows. The first scheme is shown separately in the upper part of FIG. 42 and the second scheme is shown separately in the lower part. The first scheme shows a first example of the structural analysis method of the present invention, and the second scheme shows a second example of the structural analysis method of the present invention. The wording in the figure is an example for convenience of explanation, and does not limit the present invention.
　本発明の構造解析方法の第一の例は、例えば、前記第一のスキームに示すとおり、まず、プレート１０１上に載せた被覆結晶１０２を準備する。被覆結晶１０２は、ゲルで被覆されており、本発明の結晶製造方法により製造された被覆結晶である。次に、この被覆結晶を、抗凍結剤に浸し、さらに、前述したようなループ状のマウント器具ですくい取る。すくい取った前記被覆結晶を、低温ガス吹きつけ装置等により凍結し（凍結工程）、その凍結結晶を、Ｘ線結晶構造解析または中性子線結晶構造解析等により構造解析する。このようにして、本発明の構造解析方法の第一の例を実施することができる。この第一の例は、例えば、前記ループ状のマウント器具を手作業で用いて前記被覆結晶をすくい取り、凍結させることができる。なお、前記ループ状のマウント器具は、例えば「クライオループ」などということがある。 In the first example of the structural analysis method of the present invention, for example, as shown in the first scheme, first, a coated crystal 102 placed on a plate 101 is prepared. The coated crystal 102 is coated with a gel and is a coated crystal manufactured by the crystal manufacturing method of the present invention. Next, the coated crystal is immersed in an anti-freezing agent and further scooped with a loop-shaped mounting device as described above. The coated crystal thus picked up is frozen by a low-temperature gas spraying device or the like (freezing step), and the frozen crystal is subjected to structural analysis by X-ray crystal structure analysis or neutron beam crystal structure analysis. In this way, the first example of the structural analysis method of the present invention can be implemented. In this first example, for example, the coated crystal can be skimmed and frozen using the loop-shaped mounting device manually. The loop-shaped mounting device may be referred to as “cryo loop”, for example.
　本発明の構造解析方法の第二の例は、例えば、前記第二のスキームに示すとおり、まず、被覆結晶１０２とチューブ１０３とを準備する。被覆結晶１０２は、ゲルで被覆されており、本発明の結晶製造方法により製造された被覆結晶である。チューブ１０３は、例えば、図示のように、その根元に、チューブ１０３自体を別の器具または装置により保持するための保持部が設けられていてもよい。次に、チューブ１０３の先端を被覆結晶１０２に突き刺し、結晶が含まれた部分を、被覆しているゲルごとチューブ１０３の先端に取り込む。さらに、チューブ１０３の先端に取り込まれた結晶およびゲルだけを、例えばレーザー加工等により、被覆結晶１０２の他の部分から切り離す。そして、結晶およびゲルが取り込まれたチューブ１０３の先端を、別途の容器中に準備した抗凍結剤の中に浸す。そして、チューブ１０３の先端を上に向けて置き（マウント）、そこに取り込まれた前記被覆結晶を、低温ガス吹きつけ装置等により凍結し（凍結工程）、その凍結結晶を、Ｘ線結晶構造解析または中性子線結晶構造解析等により構造解析する。このようにして、本発明の構造解析方法の第二の例を実施することができる。この第二の例は、例えば、前記各工程を必要に応じて自動化した「オートマウント」法として行うことができる。被覆結晶１０２は、例えば図４３に示すように、別のチューブの内部に準備されていてもよい。この場合、図４３に示すとおり、前記別のチューブの中にチューブ１０３の先端を突き刺し、結晶が含まれた部分を、被覆しているゲルごとチューブ１０３の先端に取り込んでもよい。これ以降の工程は、図４２の前記第二のスキームと同様に行うことができる。 In the second example of the structural analysis method of the present invention, for example, as shown in the second scheme, first, a coated crystal 102 and a tube 103 are prepared. The coated crystal 102 is coated with a gel and is a coated crystal manufactured by the crystal manufacturing method of the present invention. For example, as shown in the drawing, the tube 103 may be provided with a holding portion for holding the tube 103 itself with another instrument or device at the base thereof. Next, the tip of the tube 103 is pierced into the coated crystal 102, and the portion containing the crystal is taken into the tip of the tube 103 together with the coated gel. Furthermore, only the crystals and gel taken in at the tip of the tube 103 are separated from other parts of the coated crystal 102 by, for example, laser processing. Then, the tip of the tube 103 in which the crystals and gel are taken is immersed in an anti-freezing agent prepared in a separate container. Then, the tube 103 is placed with the tip facing upward (mount), and the coated crystal taken therein is frozen by a low-temperature gas spraying device or the like (freezing step), and the frozen crystal is analyzed by X-ray crystal structure analysis. Alternatively, structural analysis is performed by neutron beam crystal structure analysis or the like. In this way, the second example of the structural analysis method of the present invention can be implemented. This second example can be performed, for example, as an “automount” method in which the above steps are automated as necessary. For example, as shown in FIG. 43, the coated crystal 102 may be prepared inside another tube. In this case, as shown in FIG. 43, the tip of the tube 103 may be pierced into the other tube, and the portion containing the crystal may be taken into the tip of the tube 103 together with the coated gel. The subsequent steps can be performed in the same manner as in the second scheme of FIG.
　なお、前記被覆結晶において、結晶を被覆している前記ゲルが測定ノイズの原因となるなどの支障がある場合は、あらかじめ除去することが好ましい。除去方法は特に制限されないが、例えば、前述の通り、熱可逆性ハイドロゲルを冷却する方法、フェムト秒レーザー等のレーザー光で加工する方法等がある。 It should be noted that if there is a problem in the coated crystal such that the gel covering the crystal causes measurement noise, it is preferably removed in advance. The removal method is not particularly limited. For example, as described above, there are a method of cooling a thermoreversible hydrogel, a method of processing with a laser beam such as a femtosecond laser, and the like.
［結晶化スクリーニング方法および結晶化スクリーニング装置］
　次に、本発明の結晶化スクリーニング方法は、前述の通り、生体物質の結晶化条件をスクリーニングする結晶化スクリーニング方法であって、前記生体物質の結晶を製造する結晶製造工程と、前記結晶製造工程における結晶化条件をスクリーニングするスクリーニング工程を含み、前記結晶製造工程において、本発明の結晶製造方法または本発明の凍結結晶製造方法により前記結晶を製造することを特徴とする。 [Crystalling screening method and crystallization screening apparatus]
Next, as described above, the crystallization screening method of the present invention is a crystallization screening method for screening a crystallization condition of a biological material, the crystal manufacturing step for manufacturing the biological material crystal, and the crystal manufacturing step. Including the screening step of screening for crystallization conditions in the method, wherein the crystal is produced by the crystal production method of the present invention or the frozen crystal production method of the present invention.
　本発明の結晶化スクリーニング方法によれば、前述の通り、本発明の結晶製造方法により簡便に結晶を製造できることで、結晶製造条件をさほど厳密に設定しなくても良いため、スクリーニングをも簡便に行うことができる。具体的には、例えば、前記生体物質濃度、前記生体物質と前記沈殿化剤の濃度比（混合比）等の最適条件を簡便にスクリーニングすることができる。また、本発明の凍結結晶製造方法により簡便に凍結結晶を製造できることで、凍結結晶製造条件のスクリーニングを簡便に行うことができる。より具体的には、例えば、前述の通り抗凍結剤による結晶の損傷を大幅に抑制できることにより、抗凍結剤濃度等の条件のスクリーニングを簡便にすることができる。 According to the crystallization screening method of the present invention, as described above, since the crystal can be easily manufactured by the crystal manufacturing method of the present invention, it is not necessary to set the crystal manufacturing conditions so strictly, the screening is also easy. It can be carried out. Specifically, for example, optimal conditions such as the concentration of the biological material and the concentration ratio (mixing ratio) of the biological material and the precipitating agent can be easily screened. Moreover, since the frozen crystal can be easily manufactured by the method for manufacturing a frozen crystal of the present invention, screening of the frozen crystal manufacturing condition can be easily performed. More specifically, for example, as described above, the crystal damage caused by the cryoprotectant can be greatly suppressed, so that screening of conditions such as the cryoprotectant concentration can be simplified.
　さらに、本発明の結晶化スクリーニング装置は、前述の通り、生体物質の結晶化条件をスクリーニングする結晶化スクリーニング装置であって、前記生体物質の結晶を製造する結晶製造手段と、前記生体物質の結晶化条件をスクリーニングするスクリーニング手段とを含み、前記結晶製造手段が、ゲル中で前記生体物質を結晶化させる結晶化手段を含むことを特徴とする。これら以外には、本発明の結晶化スクリーニング装置は特に制限されないが、例えば、従来の結晶化スクリーニング装置よりも構成を簡略化することも可能である。例えば、前述のような前記生体物質濃度、前記生体物質と前記沈殿化剤の濃度比（混合比）、抗凍結剤濃度等の条件のスクリーニングを簡便にできることで、それらに必要な手段を省略または簡略化することも可能である。本発明の結晶化スクリーニング方法は、どのような装置や器具を用いて行っても良いが、本発明の結晶化スクリーニング装置により行うことが好ましい。 Furthermore, as described above, the crystallization screening apparatus of the present invention is a crystallization screening apparatus for screening a crystallization condition of a biological material, the crystal manufacturing means for manufacturing the biological material crystal, and the biological material crystal. Screening means for screening crystallization conditions, wherein the crystal production means includes crystallization means for crystallizing the biological material in a gel. Other than these, the crystallization screening apparatus of the present invention is not particularly limited. For example, the configuration can be simplified as compared with the conventional crystallization screening apparatus. For example, it is possible to simplify the screening of conditions such as the concentration of the biological material, the concentration ratio of the biological material and the precipitating agent (mixing ratio), the concentration of the cryoprotectant, etc. Simplification is also possible. The crystallization screening method of the present invention may be performed using any apparatus or instrument, but is preferably performed by the crystallization screening apparatus of the present invention.
　図４４に、本発明の結晶化スクリーニング装置およびそれを用いた本発明の結晶化スクリーニング方法の例を三つ示す。前記結晶化スクリーニング方法の三つの例を、それぞれ、ハンギングドロップ（Ｈａｎｇｉｎｇ　Ｄｒｏｐ）法、シッティングドロップ（Ｓｉｔｔｉｎｇ　Ｄｒｏｐ）法、および濃度勾配法という。図４４左上は、ハンギングドロップ法に用いる装置の例であり、左下は、シッティングドロップ法に用いる装置の例であり、右上は、濃度勾配法に用いる装置の例である。 FIG. 44 shows three examples of the crystallization screening apparatus of the present invention and the crystallization screening method of the present invention using the same. Three examples of the crystallization screening method are referred to as a hanging drop method, a sitting drop method, and a concentration gradient method, respectively. The upper left of FIG. 44 is an example of an apparatus used for the hanging drop method, the lower left is an example of an apparatus used for the sitting drop method, and the upper right is an example of an apparatus used for the concentration gradient method.
　まず、図４４左上の装置について説明する。図示の通り、この装置は、蓋体２０１と、プレート２０３とを主要構成要素とする。これらは、本発明の結晶化スクリーニング装置における前記結晶化手段および結晶製造手段を構成するとともに、前記スクリーニング手段をも兼ねる。蓋体２０１は板状であり、その片面には突起部２０２が設けられており、他方の面は平らな形状をしている。突起部２０２の上面は、ゲルを載せることができるように、例えば平らな形状をしている。プレート２０３の上面には、反対側まで貫通しないウェル（穴）が形成されている。前記ウェル中には、蓋体２０１に形成された突起部２０２をはめ込むことが可能である。なお、蓋体２０１に設けられた突起部２０２およびプレート２０３に形成されたウェルの数は任意であり、それぞれ１つでも複数でも良いが、スクリーニングを効率的に行うために十分な数を設けることが好ましい。また、突起部２０２は、例えば、蓋体２０１本体から取り外し可能であると、結晶の採取等の操作が行いやすいため好ましい。なお、この装置において、各構成要素の形成材料は特に制限されない。例えば、ガラス、プラスチック等、理化学機器に一般に用いられる材質であって、結晶の製造およびスクリーニングの妨げにならない材質を用いれば良い。また、各構成要素の大きさも特に制限されないが、例えば、装置全体として、実験台の上での取り扱いに便利な程度の大きさであることが好ましい。 First, the apparatus at the upper left of FIG. 44 will be described. As shown in the figure, this apparatus includes a lid 201 and a plate 203 as main components. These constitute the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serve as the screening means. The lid 201 has a plate shape, and a projection 202 is provided on one surface thereof, and the other surface has a flat shape. The upper surface of the protrusion 202 has, for example, a flat shape so that a gel can be placed thereon. A well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 203. A projection 202 formed on the lid 201 can be fitted into the well. The number of wells formed on the protrusion 202 provided on the lid 201 and the plate 203 is arbitrary, and one or more wells may be provided, but a sufficient number is provided for efficient screening. Is preferred. In addition, it is preferable that the protrusion 202 be removable from the lid 201 main body, for example, because an operation such as collecting a crystal can be easily performed. In this apparatus, the material for forming each component is not particularly limited. For example, a material that is generally used for physics and chemistry equipment, such as glass and plastic, and that does not interfere with crystal production and screening may be used. Also, the size of each component is not particularly limited, but for example, it is preferable that the entire device is of a size convenient for handling on an experimental table.
　この装置を用いた本発明の結晶化スクリーニング方法（ハンギングドロップ法）は、例えば、以下のようにして行うことができる。すなわち、まず、蓋体２０１を、突起部２０２を上に向けて準備する。次に、突起部２０２上面に、生体物質を含むゲルを載せるか、または、生体物質を含む溶液として突起部２０２上面に載せ、そこで固化（ゲル化）させる。このゲルの製造方法は、例えば前述の通りである。なお、図中では「タンパク質とゲルの混合物」と記載しているが、タンパク質に限定されず、どのような生体物質でもよい。一方、プレート２０３のウェル中には、沈殿化剤（沈殿剤）溶液を入れておく。沈殿化剤溶液の製造方法も、例えば前述の通りである。そして、蓋体２０１を上下反転させて突起部２０２を下に向け、突起部２０２をプレートウェル中にはめ込む。これにより、生体物質を含む前記ゲルを前記沈殿化剤（沈殿剤）溶液中に浸漬させ、生体物質をゲル中で結晶化させる。これにより、本発明の結晶化スクリーニング方法における結晶製造工程を行うことができる。そして、この結晶製造工程において、前記生体物質濃度および前記沈殿化剤濃度等の条件を種々変更しておき、前記結晶製造工程終了後、各条件における結晶の形成状態を観察する。これにより、結晶化条件をスクリーニングする前記スクリーニング工程を行う。ここで、突起部２０２およびウェルが複数設けられている場合は、各突起部２０２およびウェルにおいて、前記生体物質濃度および前記沈殿化剤濃度を種々変更することにより、一度の結晶製造工程で前記スクリーニング工程を行うことも可能である。以上のようにして、この装置を用いた本発明の結晶化スクリーニング方法（ハンギングドロップ法）を実施することができる。 The crystallization screening method (hanging drop method) of the present invention using this apparatus can be performed as follows, for example. That is, first, the lid 201 is prepared with the protrusion 202 facing upward. Next, a gel containing a biological material is placed on the upper surface of the protruding portion 202, or is placed on the upper surface of the protruding portion 202 as a solution containing a biological material and solidified (gelled) there. The method for producing this gel is as described above, for example. In the figure, “a mixture of protein and gel” is described. However, the present invention is not limited to protein, and any biological substance may be used. On the other hand, a precipitating agent (precipitating agent) solution is placed in the well of the plate 203. The method for producing the precipitating agent solution is also as described above, for example. Then, the lid 201 is turned upside down so that the projection 202 faces downward, and the projection 202 is fitted into the plate well. Accordingly, the gel containing the biological material is immersed in the precipitant (precipitating agent) solution, and the biological material is crystallized in the gel. Thereby, the crystal manufacturing process in the crystallization screening method of the present invention can be performed. In this crystal manufacturing process, various conditions such as the biological substance concentration and the precipitating agent concentration are changed, and after the crystal manufacturing process, the crystal formation state under each condition is observed. Thus, the screening step of screening for crystallization conditions is performed. Here, in the case where a plurality of protrusions 202 and wells are provided, the screening can be performed in a single crystal manufacturing process by variously changing the biological material concentration and the precipitating agent concentration in each protrusion 202 and well. It is also possible to carry out the process. As described above, the crystallization screening method (hanging drop method) of the present invention using this apparatus can be carried out.
　次に、図４４左下の装置について説明する。図示の通り、この装置は、プレート２０７を主要構成要素とする。このプレート２０７は、本発明の結晶化スクリーニング装置における前記結晶化手段および結晶製造手段を構成するとともに、前記スクリーニング手段をも兼ねる。プレート２０７の上面には、反対側まで貫通しないウェル（穴）が形成されている。前記ウェルの数は任意であり、１つでも複数でも良いが、スクリーニングを効率的に行うために十分な数を設けることが好ましい。なお、この装置の形成材料および大きさについては、前述の図４４左上の装置と同様である。 Next, the apparatus at the lower left of FIG. 44 will be described. As shown, this apparatus has a plate 207 as a main component. The plate 207 constitutes the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serves as the screening means. A well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 207. The number of the wells is arbitrary and may be one or more, but it is preferable to provide a sufficient number for efficient screening. Note that the forming material and the size of this device are the same as those in the upper left of FIG.
　この装置を用いた本発明の結晶化スクリーニング方法（シッティングドロップ法）は、例えば、以下のようにして行うことができる。すなわち、まず、プレート２０７のウェル中に、生体物質を含むゲルを入れるか、または、生体物質溶液の状態で前記ウェル中にいれ、そこで固化（ゲル化）させる。このゲルの製造方法は、例えば前述の通りである。図中では「タンパク質とゲルの混合物」と記載しているが、タンパク質に限定されず、どのような生体物質でもよい。次に、前記ウェル中に、沈殿化剤（沈殿剤）溶液を注入する。沈殿化剤溶液の製造方法も、例えば前述の通りである。これにより、生体物質を含む前記ゲルを前記沈殿化剤（沈殿剤）溶液中に浸漬させ、生体物質をゲル中で結晶化させる。なお、このとき、前記ウェル中への異物の混入、沈殿化剤溶液の揮発等を防ぐため、例えば、プレート２０７の上面にシール等を設置することが好ましい。これにより、本発明の結晶化スクリーニング方法における結晶製造工程を行うことができる。そして、この結晶製造工程において、前記生体物質濃度および前記沈殿化剤濃度等の条件を種々変更しておき、前記結晶製造工程終了後、各条件における結晶の形成状態を観察する。これにより、結晶化条件をスクリーニングする前記スクリーニング工程を行う。ここで、前記ウェルが複数設けられている場合は、各ウェルにおいて、前記生体物質濃度および前記沈殿化剤濃度を種々変更することにより、一度の結晶製造工程で前記スクリーニング工程を行うことも可能である。以上のようにして、この装置を用いた本発明の結晶化スクリーニング方法（シッティングドロップ法）を実施することができる。 The crystallization screening method (sitting drop method) of the present invention using this apparatus can be performed, for example, as follows. That is, first, a gel containing a biological material is placed in the well of the plate 207 or placed in the well in the state of a biological material solution and solidified (gelled) there. The method for producing this gel is as described above, for example. Although it is described as “a mixture of protein and gel” in the drawing, it is not limited to protein, and any biological material may be used. Next, a precipitant (precipitant) solution is injected into the well. The method for producing the precipitating agent solution is also as described above, for example. Accordingly, the gel containing the biological material is immersed in the precipitant (precipitating agent) solution, and the biological material is crystallized in the gel. At this time, for example, a seal or the like is preferably provided on the upper surface of the plate 207 in order to prevent foreign substances from entering the well, volatilization of the precipitant solution, and the like. Thereby, the crystal manufacturing process in the crystallization screening method of the present invention can be performed. In this crystal manufacturing process, various conditions such as the biological substance concentration and the precipitating agent concentration are changed, and after the crystal manufacturing process, the crystal formation state under each condition is observed. Thus, the screening step of screening for crystallization conditions is performed. Here, when a plurality of wells are provided, the screening step can be performed in a single crystal manufacturing step by variously changing the biological material concentration and the precipitating agent concentration in each well. is there. As described above, the crystallization screening method (sitting drop method) of the present invention using this apparatus can be carried out.
　さらに、図４４右上の装置について説明する。図示の通り、この装置は、蓋体２０９と、プレート２０６とを主要構成要素とする。これらは、本発明の結晶化スクリーニング装置における前記結晶化手段および結晶製造手段を構成するとともに、前記スクリーニング手段をも兼ねる。蓋体２０９は板状であり、その片面の一部が突出してチューブ２０５を形成している。チューブ２０５の内部は空洞であり、蓋体２０９本体に接した側の端は開口している。チューブ２０５の他端（以下「突端」という）は閉じており、例えば半透膜（透析膜）等が張られていることにより、固形物は透過せず、一定の大きさ以下の粒子のみが透過可能である。プレート２０６の上面には、反対側まで貫通しないウェル（穴）が形成されている。前記ウェル中には、蓋体２０９に形成されたチューブ２０５をはめ込むことが可能である。蓋体２０９に形成されたチューブ２０５およびプレート２０６に形成されたウェルの数は任意であり、それぞれ１つでも複数でも良いが、スクリーニングを効率的に行うために十分な数を設けることが好ましい。なお、この装置の形成材料および大きさについては、チューブ２０５端部の前記半透膜（透析膜）等以外は、前述の図４４左上の装置と同様である。 Furthermore, the apparatus in the upper right of FIG. 44 will be described. As shown in the figure, this apparatus has a lid 209 and a plate 206 as main components. These constitute the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serve as the screening means. The lid 209 has a plate shape, and a part of one surface thereof protrudes to form a tube 205. The inside of the tube 205 is hollow, and the end on the side in contact with the lid 209 main body is open. The other end of the tube 205 (hereinafter referred to as “protruding end”) is closed, and, for example, a semipermeable membrane (dialysis membrane) or the like is stretched, so that solid matter does not permeate, and only particles having a certain size or less. It can be transmitted. A well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 206. A tube 205 formed on the lid 209 can be fitted into the well. The number of wells formed on the tube 205 and the plate 206 formed on the lid 209 is arbitrary, and may be one or more, but it is preferable to provide a sufficient number for efficient screening. The forming material and the size of this device are the same as those in the upper left of FIG. 44 except for the semipermeable membrane (dialysis membrane) at the end of the tube 205.
　この装置を用いた本発明の結晶化スクリーニング方法（濃度勾配法）は、例えば、以下のようにして行うことができる。すなわち、まず、蓋体２０９に形成されたチューブ２０５内部に、生体物質を含むゲルを入れる。このゲルの製造方法は、例えば前述の通りである。前記ゲルは、ゲルの状態にしてからチューブ２０５内部に注入してもよいが、溶液（ゾル）の状態でチューブ２０５内部に注入し、チューブ２０５内部でゲル化させることが簡便で好ましい。なお、図中では「タンパク質とゲルの混合物」と記載しているが、タンパク質に限定されず、どのような生体物質でもよい。一方、プレート２０６のウェル中には、沈殿化剤（沈殿剤）溶液を入れておく。沈殿化剤溶液の製造方法も、例えば前述の通りである。そして、チューブ２０５をプレート２０６のウェル中にはめ込む。これにより、沈殿化剤が、チューブ２０５突端を通じて、前記ゲル中に浸透する。前述の通り、チューブ２０５突端には、半透膜（透析膜）等が張られていることにより、前記ゲルは透過しない。これにより、生体物質をゲル中で結晶化させる。このとき、前記ウェル中への異物の混入、ゲルの乾燥等を防ぐため、例えば、蓋体２０９の上面にシール等を設置することが好ましい。これにより、本発明の結晶化スクリーニング方法における結晶製造工程を行うことができる。このとき、前述のように、沈殿化剤は、チューブ２０５突端を通じて前記ゲル中に浸透するため、前記ゲル中において、沈殿化剤（沈殿剤）濃度に勾配が形成される。すなわち、チューブ２０５突端に近い側では沈殿化剤濃度が高くなり、前記突端から遠ざかるほど沈殿化剤濃度が低くなる。この濃度勾配が形成されることによって、チューブ２０５内部の各部で前記生体物質濃度と前記沈殿化剤濃度との比が種々変化する。このため、前記結晶製造工程終了後、チューブ２０５内部における結晶の形成状態を観察することで、結晶化条件をスクリーニングする前記スクリーニング工程を行うことができるのである。さらに、チューブ２０５およびウェルが複数設けられている場合は、各チューブ２０５およびウェルにおいて、前記生体物質濃度および前記沈殿化剤濃度を種々変更することにより、さらに詳細に前記スクリーニング工程を行うことも可能である。このようにして、この装置を用いた本発明の結晶化スクリーニング方法（濃度勾配法）を実施することができる。 The crystallization screening method (concentration gradient method) of the present invention using this apparatus can be performed, for example, as follows. That is, first, a gel containing a biological material is placed inside the tube 205 formed on the lid 209. The method for producing this gel is as described above, for example. The gel may be injected into the tube 205 after being in a gel state, but it is preferable that the gel is injected into the tube 205 in a solution (sol) state and gelled in the tube 205. In the figure, “a mixture of protein and gel” is described. However, the present invention is not limited to protein, and any biological substance may be used. On the other hand, a precipitant (precipitant) solution is placed in the well of the plate 206. The method for producing the precipitating agent solution is also as described above, for example. Then, the tube 205 is fitted into the well of the plate 206. Thereby, the precipitating agent penetrates into the gel through the tip of the tube 205. As described above, the gel 205 does not permeate due to a semipermeable membrane (dialysis membrane) or the like stretched around the protruding end of the tube 205. This causes the biological material to crystallize in the gel. At this time, for example, a seal or the like is preferably installed on the upper surface of the lid 209 in order to prevent foreign matter from entering the well, drying of the gel, and the like. Thereby, the crystal manufacturing process in the crystallization screening method of the present invention can be performed. At this time, as described above, since the precipitating agent penetrates into the gel through the tip of the tube 205, a gradient is formed in the concentration of the precipitating agent (precipitating agent) in the gel. That is, the concentration of the precipitating agent increases on the side closer to the tip of the tube 205, and the concentration of the precipitating agent decreases as the distance from the tip increases. By forming this concentration gradient, the ratio between the concentration of the biological material and the concentration of the precipitating agent varies in various parts inside the tube 205. For this reason, after the completion of the crystal production process, the screening process for screening the crystallization conditions can be performed by observing the crystal formation state in the tube 205. Further, when a plurality of tubes 205 and wells are provided, the screening step can be performed in more detail by changing the biological material concentration and the precipitant concentration in each tube 205 and well. It is. Thus, the crystallization screening method (concentration gradient method) of the present invention using this apparatus can be carried out.
　なお、濃度勾配法による結晶化スクリーニング方法は、例えば、後述の実施例のように、遠心分離装置を用いて行うこともできる。生体物質およびゲル化剤の種類および濃度、等の各種条件は、後述の実施例に限定されず、任意に設定できる。具体的には、例えば、他の実施形態と同様である。遠心分離速度等も特に制限されず、適宜設定可能である。また、濃度勾配法による結晶化スクリーニング方法は、沈殿化剤濃度の勾配を形成して行うのみならず、例えば、生体物質の濃度勾配を形成して行うこともできる。具体的には、例えば、前記生体物質を含まないゲルをあらかじめ準備しておき、そのゲルの片面から前記生体物質溶液を徐々に浸透させることで、前記ゲル中に前記生体物質濃度の勾配を形成しても良い。前記ゲルは、例えば、あらかじめ沈殿化剤を含んでいても良いし、含んでいなくても良い。 In addition, the crystallization screening method by the concentration gradient method can also be performed using a centrifuge, for example, as in Examples described later. Various conditions such as the types and concentrations of the biological material and the gelling agent are not limited to the examples described later, and can be arbitrarily set. Specifically, it is the same as that of other embodiment, for example. The centrifugation speed and the like are not particularly limited and can be set as appropriate. Moreover, the crystallization screening method by the concentration gradient method can be performed not only by forming a precipitant concentration gradient but also by forming a biological material concentration gradient, for example. Specifically, for example, a gel containing no biological material is prepared in advance, and the biological material solution is gradually permeated from one side of the gel to form a gradient of the biological material concentration in the gel. You may do it. The gel may or may not contain a precipitating agent in advance, for example.
　さらに、本発明の結晶化スクリーニング装置および結晶化スクリーニング方法は、これらに限定されず、どのような装置および方法でもよい。例えば、結晶化スクリーニング方法の各種条件は、前述の説明に限定されず、適宜変更が可能である。例えば、結晶化の方法として、図４４を用いた前述の説明では、前記生体物質溶液を含むゲルを沈殿化剤溶液に接触もしくは近接させる方法を示した。しかし、前述の通り、沈殿化剤を用いた結晶化方法は、例えば、前記生体物質溶液にゲル化剤とともに沈殿化剤を混合しておき、ゲル化させて沈殿化剤を含むゲルとし、ゲル化後そのまま静置して結晶を析出させる方法でもよい。また、結晶化スクリーニング装置としては、例えば、図４５に示すように、前記「濃度勾配法」の装置（図４４右上に示す装置）のチューブ２０５を、浅いウェル３０５に変えた装置でもよい。図４５において、蓋体３０４およびプレート３０６は、ウェルの深さがウェル３０５に合わせて浅い以外は、前記「濃度勾配法」の装置（図４４右上に示す装置）と同様である。この装置を用いた結晶化スクリーニング方法は、ゲル中における濃度勾配形成がない以外は、前記「濃度勾配法」の装置と同様であり、あるいは図４４に示した他の装置と同様である。図４５の装置は、蓋体３０４およびプレート３０６に形成されたウェルが浅いことにより、前記「濃度勾配法」の装置と比較して操作全般が簡便であるという利点がある。また、図４５の装置は、ゲルと沈殿化剤溶液とがそれぞれ別のウェル中に存在するため、前記「ハンギングドロップ法」および前記「シッティングドロップ法」の装置と比較すると、前記ゲルと前記沈殿化剤溶液との分離操作が簡便である利点がある。 Furthermore, the crystallization screening apparatus and the crystallization screening method of the present invention are not limited to these, and any apparatus and method may be used. For example, various conditions of the crystallization screening method are not limited to the above description, and can be changed as appropriate. For example, in the above description using FIG. 44 as a crystallization method, a method of bringing the gel containing the biological material solution into contact with or in proximity to the precipitant solution is shown. However, as described above, the crystallization method using a precipitating agent is, for example, mixing a precipitating agent together with a gelling agent in the biological material solution and gelling it to obtain a gel containing the precipitating agent. Alternatively, the crystal may be precipitated by allowing it to stand as it is. Further, as the crystallization screening apparatus, for example, as shown in FIG. 45, an apparatus in which the tube 205 of the “concentration gradient method” apparatus (apparatus shown in the upper right of FIG. 44) is replaced with a shallow well 305 may be used. 45, the lid 304 and the plate 306 are the same as the “concentration gradient method” apparatus (the apparatus shown in the upper right of FIG. 44) except that the depth of the well is shallow to match the well 305. The crystallization screening method using this apparatus is the same as the apparatus of the “concentration gradient method”, except that there is no concentration gradient formation in the gel, or is the same as the other apparatus shown in FIG. The apparatus of FIG. 45 has an advantage that the overall operation is simpler than the apparatus of the “concentration gradient method” because the wells formed in the lid 304 and the plate 306 are shallow. 45, since the gel and the precipitating agent solution are present in different wells, the gel and the precipitation are compared with the devices of the “hanging drop method” and the “sitting drop method”. There is an advantage that the separation operation from the agent solution is simple.
　なお、本発明の結晶化スクリーニング方法は、例えば、自動化して行うことが、効率的で好ましい。例えば図４６に示すように、前記生体物質の溶液（ゾル）を分注装置により自動的に各ウェルあるいはチューブ内部などに分注するなどの方法により、前記結晶製造工程を自動化してもよい。なお、同図においては「温度コントローラー付きタンパク質分注装置」と記載しているが、これは単なる一例であり、生体物質は、タンパク質に限定されずどのような物質でもよいし、分注装置も、どのような分注装置でもよい。また、例えば、前記結晶製造工程終了後、結晶の形成状態を、図示のように顕微鏡で自動的に観察および写真撮影することで、前記スクリーニング工程を自動化してもよい。 In addition, it is efficient and preferable that the crystallization screening method of the present invention is performed automatically, for example. For example, as shown in FIG. 46, the crystal manufacturing process may be automated by a method of automatically dispensing the solution (sol) of the biological material into each well or the inside of a tube by a dispensing device. In the figure, it is described as “a protein dispensing device with a temperature controller”, but this is merely an example, and the biological material is not limited to proteins, and any material may be used. Any dispensing device may be used. Further, for example, after the crystal manufacturing process is completed, the screening process may be automated by automatically observing and photographing the crystal formation state with a microscope as illustrated.
　なお、本発明の結晶化スクリーニング装置は、生体物質の結晶を製造する結晶製造手段を含んでいるため、生体物質の結晶を製造する結晶製造装置としても使用可能である。 Note that the crystallization screening apparatus of the present invention includes a crystal manufacturing means for manufacturing a crystal of biological material, and therefore can be used as a crystal manufacturing apparatus for manufacturing a crystal of biological material.
　次に、本発明の実施例および比較例について説明する。 Next, examples and comparative examples of the present invention will be described.
　以下の実施例１～７および比較例１～７では、検体（被解析物質）として、下記（１）～（３）のタンパク質結晶を用いた。実施例８以下では、検体（被解析物質）として、下記（１）～（２）および（４）～（７）のタンパク質結晶、または（８）の核酸（ＤＮＡ）結晶を用いた。
（１）リゾチーム
（２）グルコースイソメラーゼ
（３）ソーマチン
（４）エラスターゼ
（５）Ｓｙｎｅｃｈｏｃｏｃｃｕｓ由来ホスホリブロキナーゼ（ＰＲＫ）
　（Ｐｈｏｓｐｈｏｒｉｂｕｌｏｋｉｎａｓｅ（ＰＲＫ）／Ｓｙｎｅｃｈｏｃｏｃｃｕｓ）
（６）セリンアセチルトランスフェラーゼ（ＳＡＴ）
（７）大腸菌異物排出トランスポーター（ＡｃｒＢ）
（８）ＤＮＡ In Examples 1 to 7 and Comparative Examples 1 to 7 below, protein crystals of the following (1) to (3) were used as specimens (analyzed substances). In Examples 8 and below, the following (1) to (2) and (4) to (7) protein crystals or (8) nucleic acid (DNA) crystals were used as specimens (analytes).
(1) Lysozyme (2) Glucose isomerase (3) Saumatine (4) Elastase (5) Synechococcus-derived phosphoribrokinase (PRK)
(Phosphoribokinase (PRK) / Synechococcus)
(6) Serine acetyltransferase (SAT)
(7) E. coli foreign body excretion transporter (AcrB)
(8) DNA
　Ｘ線源は、株式会社リガク製の商品名Ｕｌｔｒａｘ－１８（Ｃｕ対陰極）を用いた。検出器は、株式会社リガク製のＲ－ＡＸＩＳ　ＩＶ＋＋を用いた。測定電圧は５０ｋＶ、測定電流は１００ｍＡ、ビーム径は０．３ｍｍとした。 The trade name Ultrax-18 (Cu counter cathode) manufactured by Rigaku Corporation was used as the X-ray source. The detector used was R-AXIS IV ++ manufactured by Rigaku Corporation. The measurement voltage was 50 kV, the measurement current was 100 mA, and the beam diameter was 0.3 mm.
［実施例１］
　アガロース－III（（株）同仁化学研究所製、ゲル化温度は約３７～３９℃）２質量％と水９８質量％を加熱溶解させたもの（Ａ）０．１ｍＬとニワトリ卵白リゾチーム溶液（Ｂ）０．１ｍＬの両者を３５℃の一定温度下で混合し結晶化溶液（Ｃ）とした。この結晶化溶液（Ｃ）が固化したゲルとなる前に、キャピラリー（ヒルゲンベルグ社製）へ充填した。前記キャピラリーは、ガラス製（８０ｍｍ、内径０．７ｍｍ）であり、内部の管状空間に固化する前の結晶化溶液（Ｃ）を充填した。この結晶化溶液（Ｃ）は室温にて数十秒後に固化し、キャピラリー内部空間は結晶化溶液（Ｃ）が固化したゲルで充満された。次に、充填されたキャピラリーを、沈殿化剤溶液の約２ｍｌが注入された試験管容器に挿入した。この試験管容器は、直径約１６ｍｍ、長さ約１３３ｍｍ、のガラス製を用いた。このとき、キャピラリーの挿入端において、固化（ゲル化）した結晶化溶液（Ｃ）と沈殿化剤溶液との接触界面が形成された。このとき、固化した結晶化溶液（Ｃ）と沈殿化剤溶液との間に気泡が入らないように注意した。さらに、試験管の開口端を栓によって密栓した。なお、以下に、前記タンパク質溶液および沈殿化剤溶液の組成を示す。 [Example 1]
Agarose-III (manufactured by Dojindo Laboratories Co., Ltd., gelation temperature is about 37-39 ° C.) 2% by mass and 98% by mass of water dissolved by heating (A) 0.1 mL and chicken egg white lysozyme solution (B ) 0.1 mL of both were mixed at a constant temperature of 35 ° C. to obtain a crystallization solution (C). Before the crystallization solution (C) became a solidified gel, it was filled into a capillary (manufactured by Hirgenberg). The capillary was made of glass (80 mm, inner diameter 0.7 mm), and filled with a crystallization solution (C) before solidifying into an internal tubular space. The crystallization solution (C) solidified after several tens of seconds at room temperature, and the capillary inner space was filled with the gel in which the crystallization solution (C) was solidified. The filled capillary was then inserted into a test tube container into which approximately 2 ml of the precipitant solution was injected. This test tube container was made of glass having a diameter of about 16 mm and a length of about 133 mm. At this time, a contact interface between the solidified (gelled) crystallization solution (C) and the precipitating agent solution was formed at the insertion end of the capillary. At this time, care was taken to prevent bubbles from entering between the solidified crystallization solution (C) and the precipitant solution. Furthermore, the open end of the test tube was sealed with a stopper. In addition, the composition of the protein solution and the precipitating agent solution is shown below.
タンパク質溶液（Ｂ）：
３０mg/mLニワトリ卵白リゾチーム
０.１M　酢酸バッファーｐH４．５
 
沈殿化剤溶液：
１.０M塩化ナトリウム
０.１M酢酸バッファーｐH４．５
 
結晶化温度：
２０℃ Protein solution (B):
30 mg / mL chicken egg white lysozyme 0.1 M acetate buffer pH 4.5

Precipitating agent solution:
1.0M sodium chloride 0.1M acetate buffer pH 4.5

Crystallization temperature:
20 ° C
　前記キャピラリーを挿入し、栓によって密封した試験管を、結晶化温度２０℃でセットアップ後、数日内に、固化したゲル中にリゾチーム結晶を確認した。試験管からキャピラリーを取り出し、キャピラリー内の固化したゲル中に晶出したリゾチーム結晶を以下の方法で取り出した。すなわち、まず、ガラスプレートを準備し、その上に沈殿化剤溶液と抗凍結剤溶液（本実施例では１００％ジメチルスルホキシド溶液）をそれぞれ約３０μＬ、別々の位置に分注してのせておいた。ピンセットやキャピラリーカッター等を用いて取り出したい結晶の前後５～１０ｍｍの範囲でキャピラリーを切断または破砕し、次に固化したゲルをカッター等で切断した。さらに、取り出したゲル中にある結晶を、先に準備しておいた沈殿化剤溶液中に移動させた。マイクロツール（ハンプトンリサーチ社製）などを用いて顕微鏡下にて、リゾチーム結晶の周りにあるゲルを厚さ約０．１ｍｍになるまで慎重に剥ぎ取った。厚さ約０．１ｍｍのゲルに覆われたリゾチーム結晶をループ状のマウント器具を用いて沈殿化剤溶液からすくい上げ、先に準備しておいた抗凍結剤溶液に数秒間浸漬した後、素早く取り出し凍結した。さらにＸ線結晶構造解析を行った。Ｘ線回折像撮影は、レンズと結晶との距離１００ｍｍ、露光時間１ｍｉｎ、振動角Δ１°、撮影枚数２０枚で行った。その結果、リゾチームの結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 After setting up the test tube in which the capillary was inserted and sealed with a stopper at a crystallization temperature of 20 ° C., lysozyme crystals were confirmed in the solidified gel within a few days. The capillary was taken out from the test tube, and the lysozyme crystal crystallized in the solidified gel in the capillary was taken out by the following method. That is, first, a glass plate was prepared, and about 30 μL each of a precipitating agent solution and an anti-freezing agent solution (100% dimethyl sulfoxide solution in this example) were dispensed on different positions. . The capillary was cut or crushed within a range of 5 to 10 mm before and after the crystal to be taken out using tweezers or a capillary cutter, and then the solidified gel was cut with a cutter or the like. Furthermore, the crystals in the taken-out gel were moved into the precipitating agent solution prepared in advance. Using a micro tool (manufactured by Hampton Research) or the like, the gel around the lysozyme crystal was carefully peeled off to a thickness of about 0.1 mm under a microscope. A lysozyme crystal covered with a gel with a thickness of about 0.1 mm is scooped up from the precipitant solution using a loop-shaped mounting device, immersed in the previously prepared antifreeze solution for several seconds, and then quickly removed. Frozen. Further, X-ray crystal structure analysis was performed. X-ray diffraction image photography was performed at a distance of 100 mm between the lens and the crystal, an exposure time of 1 min, a vibration angle Δ1 °, and 20 shots. As a result, the crystal of lysozyme could be easily captured without damage, and it was resistant to liquid nitrogen treatment without being damaged by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（リゾチーム結晶：実施例１）
Ｐ４３２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝ｂ＝７７．２Å，　ｃ＝３７．３Å
α＝β＝γ＝９０°
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０４３（）
Ｍｏｓａｉｃｉｔｙ　０．６７° (Lysozyme crystal: Example 1)
P43212
Resolution 50.00-1.64 ()
a = b = 77.2Å, c = 37.3Å
α = β = γ = 90 °
I / σ (I) ()
Rmerge 0.043 ()
Mosaicity 0.67 °
　図１Ａおよび図１Ｂに、ゲルで保護された実施例１のリゾチーム結晶を、それぞれ撮影角度を変えて撮影した写真を示す。図示の通り、実施例１では、大きく高品質なリゾチーム単結晶が得られた。また、図２Ａおよび図２Ｂに、実施例１のリゾチーム結晶のＸ線回折像を示す。図２Ａは全体図であり、図２Ｂは、一部（図２Ａ右下の枠線部分）の拡大図である。図示の通り、良好なＸ線回折像が得られたことが分かる。 FIG. 1A and FIG. 1B show photographs of the lysozyme crystals of Example 1 protected with a gel, taken at different angles. As shown in the figure, in Example 1, a large and high quality lysozyme single crystal was obtained. 2A and 2B show X-ray diffraction images of the lysozyme crystal of Example 1. FIG. FIG. 2A is an overall view, and FIG. 2B is an enlarged view of a part (the frame line portion at the lower right of FIG. 2A). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
［実施例２］
　ニワトリ卵白リゾチーム溶液に代えてグルコースイソメラーゼ溶液を用い、タンパク質溶液及び沈殿化剤溶液の組成、抗凍結剤の種類以外は実施例１と同様にして結晶を製造した。結晶化温度４℃でセットアップ後、約１週間で、固化したゲル中にグルコースイソメラーゼ結晶を確認した。以下に、タンパク質溶液および沈殿化剤溶液の組成を示す。 [Example 2]
A glucose isomerase solution was used in place of the chicken egg white lysozyme solution, and crystals were produced in the same manner as in Example 1 except for the composition of the protein solution and the precipitating agent solution and the type of the cryoprotectant. About 1 week after setup at a crystallization temperature of 4 ° C., glucose isomerase crystals were confirmed in the solidified gel. The composition of the protein solution and the precipitating agent solution is shown below.
タンパク質溶液：
２５mg/mLグルコースイソメラーゼ
０.１M　ヘペスバッファー　pH　７．５
１ｍM　塩化マグネシウム
 
沈殿化剤溶液：
２.５M　硫酸アンモニウム
０.１M　ヘペスバッファー　pH７．５
 
結晶化温度：
４℃ Protein solution:
25 mg / mL glucose isomerase 0.1 M Hepes buffer pH 7.5
1 mM magnesium chloride
Precipitating agent solution:
2.5M ammonium sulfate 0.1M Hepes buffer pH7.5

Crystallization temperature:
4 ℃
　抗凍結剤溶液は、本実施例では２．５M酢酸リチウム溶液を用いてＸ線結晶構造解析を行った。その結果、グルコースイソメラーゼ結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 In this example, the antifreezing agent solution was subjected to X-ray crystal structure analysis using a 2.5M lithium acetate solution. As a result, the glucose isomerase crystal could be easily captured without damage, and it was resistant to liquid nitrogen treatment without being damaged by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（グルコースイソメラーゼ結晶：実施例２）
Ｐ２１２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝８５．９Å，ｂ＝９４．１Å，　ｃ＝９８．３Å
α＝β＝γ＝９０°
Ｕｎｉｑｕｅ　ｒｅｆｌｅｃｔｉｏｎｓ　
Ｃｏｍｐｌｅｔｅｎｅｓｓ　
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０３８（）
Ｍｏｓａｉｃｉｔｙ　０．６４° (Glucose isomerase crystal: Example 2)
P21212
Resolution 50.00-1.64 ()
a = 85.9Å, b = 94.1Å, c = 98.3Å
α = β = γ = 90 °
Unique reflections
Completeness
I / σ (I) ()
Rmerge 0.038 ()
Mosaicity 0.64 °
　図３Ａ、図３Ｂ、図４、図５Ａおよび図５Ｂに、実施例２のグルコースイソメラーゼ結晶のＸ線回折像を示す。図３Ａは全体図であり、図３Ｂは、一部（図３Ａ右上の枠線部分）の拡大図である。図４は、条件を変えて撮影したＸ線回折像である。図５Ａは、さらに条件を変えて測定したＸ線回折像の全体図であり、図５Ｂは、一部（図５Ａ右下の枠線部分）の拡大図である。図示の通り、良好なＸ線回折像が得られたことが分かる。また、図６Ａ～Ｄに、実施例２のリゾチーム結晶を示す。図６Ａは、抗凍結剤溶液に浸漬する直前の状態を示す図であり、単結晶が得られている。図６Ｂは、抗凍結剤溶液に浸漬してから５秒後、図６Ｃは、浸漬してから１分後、図６Ｄは、浸漬してから５分後の状態をそれぞれ示す。図Ａ～Ｄ右側の結晶は、抗凍結剤溶液浸漬前から若干の欠陥があったため、浸漬後次第に崩壊したが、左側の結晶は、良好な単結晶であり、５分間浸漬しても全く崩壊しなかった。 3A, 3B, 4, 5A and 5B show X-ray diffraction images of the glucose isomerase crystal of Example 2. FIG. FIG. 3A is an overall view, and FIG. 3B is an enlarged view of a part (the frame line portion at the upper right of FIG. 3A). FIG. 4 is an X-ray diffraction image taken under different conditions. FIG. 5A is an overall view of an X-ray diffraction image measured by further changing the conditions, and FIG. 5B is an enlarged view of a part (the frame line part at the lower right of FIG. 5A). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained. 6A to 6D show the lysozyme crystal of Example 2. FIG. FIG. 6A is a diagram showing a state immediately before dipping in the antifreezing agent solution, and a single crystal is obtained. FIG. 6B shows a state after 5 seconds from the immersion in the cryoprotectant solution, FIG. 6C shows a state after 1 minute from the immersion, and FIG. 6D shows a state after 5 minutes after the immersion. The crystals on the right side of FIGS. A to D collapsed gradually after immersion because they had some defects before immersion in the cryoprotectant solution, but the crystals on the left side were good single crystals and were completely disintegrated even after being immersed for 5 minutes. I did not.
［実施例３］
　グルコースイソメラーゼ溶液を用い、抗凍結剤の種類以外は実施例２と同様にして結晶化を行った。セットアップ後、約１週間で固化したゲル中にグルコースイソメラーゼ結晶を確認した。抗凍結剤溶液は、本実施例では１００％グリセロールを用いてＸ線結晶構造解析を行った。その結果、グルコースイソメラーゼ結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 [Example 3]
Using a glucose isomerase solution, crystallization was performed in the same manner as in Example 2 except for the type of anti-freezing agent. After the setup, glucose isomerase crystals were confirmed in the gel solidified in about one week. In this example, the cryoprotectant solution was subjected to X-ray crystal structure analysis using 100% glycerol. As a result, the glucose isomerase crystal could be easily captured without damage, and it was resistant to liquid nitrogen treatment without being damaged by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（グルコースイソメラーゼ結晶：実施例３）
Ｐ２１２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝８５．９Å，ｂ＝９２．２Å，　ｃ＝９９．０Å
α＝β＝γ＝９０°
Ｕｎｉｑｕｅ　ｒｅｆｌｅｃｔｉｏｎｓ　
Ｃｏｍｐｌｅｔｅｎｅｓｓ　
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０７７（）
Ｍｏｓａｉｃｉｔｙ　０．７１° (Glucose isomerase crystal: Example 3)
P21212
Resolution 50.00-1.64 ()
a = 85.9Å, b = 92.2Å, c = 99.0Å
α = β = γ = 90 °
Unique reflections
Completeness
I / σ (I) ()
Rmerge 0.077 ()
Mosaicity 0.71 °
［実施例４］
グルコースイソメラーゼ溶液を用い、抗凍結剤の種類以外は実施例２と同様にして結晶化を行った。セットアップ後、約１週間で固化したゲル中にグルコースイソメラーゼ結晶を確認した。抗凍結剤溶液は、本実施例では５０％グリセロール溶液を用いてＸ線結晶構造解析を行った。その結果、グルコースイソメラーゼ結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 [Example 4]
Using a glucose isomerase solution, crystallization was performed in the same manner as in Example 2 except for the type of anti-freezing agent. After the setup, glucose isomerase crystals were confirmed in the gel solidified in about one week. In this example, the antifreeze solution was analyzed by X-ray crystal structure using a 50% glycerol solution. As a result, the glucose isomerase crystal could be easily captured without damage, and it was resistant to liquid nitrogen treatment without being damaged by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（グルコースイソメラーゼ結晶：実施例４）
Ｐ２１２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝８６．０Å，ｂ＝９２．４Å，　ｃ＝９９．０Å
α＝β＝γ＝９０°
Ｖｏｌｕｍｅ　２２２３０２．０
Ｕｎｉｑｕｅ　ｒｅｆｌｅｃｔｉｏｎｓ　
Ｃｏｍｐｌｅｔｅｎｅｓｓ　
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０５１（）
Ｍｏｓａｉｃｉｔｙ　０．５１° (Glucose isomerase crystal: Example 4)
P21212
Resolution 50.00-1.64 ()
a = 86.0 ?, b = 92.4 ?, c = 99.0?
α = β = γ = 90 °
Volume 222302.0
Unique reflections
Completeness
I / σ (I) ()
Rmerge 0.051 ()
Mosaicity 0.51 °
［実施例５］
ニワトリ卵白リゾチーム溶液に代えてソーマチン溶液を用い、タンパク質溶液及び沈殿化剤溶液の組成、抗凍結剤の種類以外は実施例１と同様にして結晶化を行った。結晶化温度２０℃でセットアップ後、約１週間で固化したゲル中にグルコースイソメラーゼ結晶を確認した。以下に、タンパク質溶液及び沈殿化剤溶液の組成を示す。 [Example 5]
Crystallization was carried out in the same manner as in Example 1 except that a thaumatin solution was used in place of the chicken egg white lysozyme solution and the composition of the protein solution and the precipitating agent solution and the type of the antifreezing agent were used. After setting up at a crystallization temperature of 20 ° C., glucose isomerase crystals were confirmed in the gel solidified in about one week. Below, the composition of a protein solution and a precipitant solution is shown.
タンパク質溶液：
２０mg/mLソーマチン
０.１M　N－（２－アセトアミド）イミノ二酢酸バッファー　pH６．５
 
沈殿化剤溶液：
２.０M　酒石酸ナトリウムカリウム
０.１M　N－（２－アセトアミド）イミノ二酢酸バッファー　pH６．５
 
結晶化温度：
２０℃ Protein solution:
20 mg / mL thaumatin 0.1 M N- (2-acetamido) iminodiacetic acid buffer pH 6.5

Precipitating agent solution:
2.0 M potassium sodium tartrate 0.1 M N- (2-acetamido) iminodiacetic acid buffer pH 6.5

Crystallization temperature:
20 ° C
　抗凍結剤溶液は、本実施例では１００％グリセロールを用いてＸ線結晶構造解析を行った。その結果、ソーマチン結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 In this example, the cryoprotectant solution was subjected to X-ray crystal structure analysis using 100% glycerol. As a result, thaumatin crystals could be easily captured without damage, and withstand damage to liquid nitrogen without damage caused by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（ソーマチン結晶：実施例５）
Ｐ４１２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝ｂ＝５７．５Å，　ｃ＝１４９．８Å
α＝β＝γ＝９０°
Ｕｎｉｑｕｅ　ｒｅｆｌｅｃｔｉｏｎｓ　
Ｃｏｍｐｌｅｔｅｎｅｓｓ
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０３２（）
Ｍｏｓａｉｃｉｔｙ　０．４９° (Thomatin crystals: Example 5)
P41212
Resolution 50.00-1.64 ()
a = b = 57.5Å, c = 149.8Å
α = β = γ = 90 °
Unique reflections
Completeness
I / σ (I) ()
Rmerge 0.032 ()
Mosaicity 0.49 °
　図７Ａに、ゲルで保護された実施例５のソーマチン結晶の写真を示す。図示の通り、実施例５では、大きく高品質なソーマチン単結晶が得られた。また、図７Ｂおよび図７Ｃに、実施例５のソーマチン結晶のＸ線回折像を示す。図７Ｂは全体図であり、図７Ｃは、一部（図７Ｂ下部の枠線部分）の拡大図である。図示の通り、良好なＸ線回折像が得られたことが分かる。 FIG. 7A shows a photograph of the thaumatin crystal of Example 5 protected with a gel. As illustrated, in Example 5, a large and high quality thaumatin single crystal was obtained. 7B and 7C show X-ray diffraction images of the thaumatin crystals of Example 5. FIG. FIG. 7B is an overall view, and FIG. 7C is an enlarged view of a part (the frame line portion at the bottom of FIG. 7B). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
［実施例６］
　ソーマチン溶液を用い、抗凍結剤の種類以外は実施例５と同様にして結晶化を行った。セットアップ後、約１週間で固化したゲル中にソーマチン結晶を確認した。抗凍結剤溶液は、本実施例では１００％ポリエチレングリコール（平均分子量４００）を用いてＸ線結晶構造解析を行った。その結果、ソーマチン結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 [Example 6]
Using a thaumatin solution, crystallization was performed in the same manner as in Example 5 except for the type of the cryoprotectant. After the setup, thaumatin crystals were confirmed in the gel solidified in about one week. In this example, the cryoprotectant solution was subjected to X-ray crystal structure analysis using 100% polyethylene glycol (average molecular weight 400). As a result, thaumatin crystals could be easily captured without damage, and withstand damage to liquid nitrogen without damage caused by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（ソーマチン結晶：実施例６）
Ｐ４１２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝ｂ＝５７．９Å，　ｃ＝１５０．１Å
α＝β＝γ＝９０°
Ｕｎｉｑｕｅ　ｒｅｆｌｅｃｔｉｏｎｓ　
Ｃｏｍｐｌｅｔｅｎｅｓｓ　
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０５５（）
Ｍｏｓａｉｃｉｔｙ　０．７４° (Thomatin crystals: Example 6)
P41212
Resolution 50.00-1.64 ()
a = b = 57.9Å, c = 150.1Å
α = β = γ = 90 °
Unique reflections
Completeness
I / σ (I) ()
Rmerge 0.055 ()
Mosaicity 0.74 °
［実施例７］
　ソーマチン溶液を用い、抗凍結剤の種類以外は実施例５と同様にして結晶化を行った。セットアップ後、約１週間で固化したゲル中にソーマチン結晶を確認した。抗凍結剤溶液は、本実施例では６０％ポリエチレングリコール（平均分子量４０００）溶液を用いてＸ線結晶構造解析を行った。その結果、ソーマチン結晶を損傷なく容易に捕捉することができ、また抗凍結剤溶液による損傷も無く液体窒素処理にも耐え、良好なＸ線回折像が得られた。以下に、結晶構造解析結果を示す。なお、１Å＝１０－１０ｍである。 [Example 7]
Using a thaumatin solution, crystallization was performed in the same manner as in Example 5 except for the type of the cryoprotectant. After the setup, thaumatin crystals were confirmed in the gel solidified in about one week. In this example, the cryoprotectant solution was subjected to X-ray crystal structure analysis using a 60% polyethylene glycol (average molecular weight 4000) solution. As a result, thaumatin crystals could be easily captured without damage, and withstand damage to liquid nitrogen without damage caused by the antifreezing agent solution, and a good X-ray diffraction image was obtained. The crystal structure analysis results are shown below. Note that 1 な お = 10 −10 m.
（ソーマチン結晶：実施例７）
Ｐ４１２１２
Ｒｅｓｏｌｕｔｉｏｎ　５０．００－１．６４（）
ａ＝ｂ＝５７．７Å，　ｃ＝１５０．１Å
α＝β＝γ＝９０°
Ｕｎｉｑｕｅ　ｒｅｆｌｅｃｔｉｏｎｓ　
Ｃｏｍｐｌｅｔｅｎｅｓｓ　
Ｉ／σ（Ｉ）（）
Ｒｍｅｒｇｅ　０．０４９（）
Ｍｏｓａｉｃｉｔｙ　０．５２° (Thomatin crystals: Example 7)
P41212
Resolution 50.00-1.64 ()
a = b = 57.7 mm, c = 150.1 mm
α = β = γ = 90 °
Unique reflections
Completeness
I / σ (I) ()
Rmerge 0.049 ()
Mosaicity 0.52 °
　図８Ａに、ゲルで保護された実施例７のソーマチン結晶の写真を示す。図示の通り、実施例７では、大きく高品質なソーマチン単結晶が得られた。また、図８Ｂおよび図８Ｃに、実施例７のソーマチン結晶のＸ線回折像を示す。図８Ｂは全体図であり、図８Ｃは、一部（図８Ｂ下部の枠線部分）の拡大図である。図示の通り、良好なＸ線回折像が得られたことが分かる。 FIG. 8A shows a photograph of the thaumatin crystal of Example 7 protected with a gel. As illustrated, in Example 7, a large and high quality thaumatin single crystal was obtained. 8B and 8C show X-ray diffraction images of the thaumatin crystal of Example 7. FIG. FIG. 8B is an overall view, and FIG. 8C is an enlarged view of a part (a frame portion at the bottom of FIG. 8B). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
　なお、前記アガロース－III（（株）同仁化学研究所製、ゲル化温度は約３７～３９℃）に代えてアガロースＩＸ－Ａ（シグマ社製、ゲル化温度は約８～１７℃）を用いることと、温度条件を変更すること以外は実施例１～７と同様にして結晶および凍結結晶を製造し、同様に良好な結果が得られた。 Instead of the agarose-III (manufactured by Dojindo Laboratories, Inc., gelation temperature is about 37 to 39 ° C.), agarose IX-A (manufactured by Sigma, gelation temperature is about 8 to 17 ° C.) is used. In addition, crystals and frozen crystals were produced in the same manner as in Examples 1 to 7 except that the temperature conditions were changed, and similarly good results were obtained.
［比較例１］
　アガロースを添加しないリゾチーム溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、数日内にリゾチーム結晶を確認した。キャピラリーから取り出したリゾチーム結晶は、抗凍結剤溶液（１００％ジメチルスルホキシド溶液）に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。図９Ａおよび図９Ｂに、比較例１のリゾチーム結晶の写真を示す。図９Ａは、抗凍結剤溶液に浸漬する直前の状態を示す写真であり、単結晶が得られている。図９Ｂは、抗凍結剤溶液に浸漬した直後の状態を示す写真であり、結晶は粉々になっている。 [Comparative Example 1]
Crystallization was carried out in exactly the same manner as in Example 1, except that a lysozyme solution without addition of agarose was used. After setup, lysozyme crystals were confirmed within a few days. The lysozyme crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the antifreeze solution (100% dimethyl sulfoxide solution). Therefore, X-ray crystal structure analysis could not be performed. 9A and 9B show photographs of the lysozyme crystal of Comparative Example 1. FIG. FIG. 9A is a photograph showing a state immediately before dipping in the cryoprotectant solution, and a single crystal is obtained. FIG. 9B is a photograph showing a state immediately after being immersed in the anti-freezing agent solution, and the crystals are shattered.
［比較例２］
　アガロースを添加しないグルコースイソメラーゼ溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、約１週間でグルコースイソメラーゼ結晶を確認した。キャピラリーから取り出したグルコースイソメラーゼ結晶は、抗凍結剤溶液（２．５M酢酸リチウム溶液）に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。 [Comparative Example 2]
Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (2.5M lithium acetate solution). Therefore, X-ray crystal structure analysis could not be performed.
［比較例３］
　アガロースを添加しないグルコースイソメラーゼ溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、約１週間でグルコースイソメラーゼ結晶を確認した。キャピラリーから取り出したグルコースイソメラーゼ結晶は、抗凍結剤溶液（１００％グリセロール）に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。 [Comparative Example 3]
Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (100% glycerol). Therefore, X-ray crystal structure analysis could not be performed.
［比較例４］
　アガロースを添加しないグルコースイソメラーゼ溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、約１週間でグルコースイソメラーゼ結晶を確認した。キャピラリーから取り出したグルコースイソメラーゼ結晶は、抗凍結剤溶液（５０％グリセロール溶液）に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。 [Comparative Example 4]
Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystal taken out from the capillary was broken into pieces and melted at the moment of immersion in the antifreeze solution (50% glycerol solution). Therefore, X-ray crystal structure analysis could not be performed.
［比較例５］
　アガロースを添加しないソーマチン溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、約１週間でソーマチン結晶を確認した。キャピラリーから取り出したソーマチン結晶は、抗凍結剤溶液（１００％グリセロール）に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。 [Comparative Example 5]
Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (100% glycerol). Therefore, X-ray crystal structure analysis could not be performed.
［比較例６］
　アガロースを添加しないソーマチン溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、約１週間でソーマチン結晶を確認した。キャピラリーから取り出したソーマチン結晶は、抗凍結剤溶液（１００％ポリエチレングリコール（平均分子量４００））に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。図１０Ａおよび図１０Ｂに、比較例６のリゾチーム結晶の写真を示す。図１０Ａは、抗凍結剤溶液に浸漬する直前の状態を示す写真であり、単結晶が得られている。図１０Ｂは、抗凍結剤溶液に浸漬した直後の状態を示す写真であり、結晶は粉々になっている。 [Comparative Example 6]
Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and dissolved at the moment of immersion in an anti-freezing agent solution (100% polyethylene glycol (average molecular weight 400)). Therefore, X-ray crystal structure analysis could not be performed. 10A and 10B show photographs of the lysozyme crystal of Comparative Example 6. FIG. FIG. 10A is a photograph showing a state immediately before dipping in an anti-freezing agent solution, and a single crystal is obtained. FIG. 10B is a photograph showing a state immediately after being immersed in the cryoprotectant solution, and the crystals are shattered.
［比較例７］
　アガロースを添加しないソーマチン溶液を用いる以外は実施例１と全く同様に結晶化を行った。セットアップ後、約１週間でソーマチン結晶を確認した。キャピラリーから取り出したソーマチン結晶は、抗凍結剤溶液（６０％ポリエチレングリコール（平均分子量４０００）溶液）に浸漬した瞬間に粉々に割れ、溶けてしまった。そのため、Ｘ線結晶構造解析を行うことができなかった。 [Comparative Example 7]
Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in an anti-freezing agent solution (60% polyethylene glycol (average molecular weight 4000) solution). Therefore, X-ray crystal structure analysis could not be performed.
　以上の通り、実施例１～７によれば、ゲル中で得られた３種類のタンパク質結晶（リゾチーム、グルコースイソメラーゼ、ソーマチン）は、全て、抗凍結剤による結晶への損傷がほぼ完全に防止された。すなわち、ゲルでコートされた実施例のタンパク質結晶と、比較例のフリーな結晶を比べると、３種のタンパク質結晶ともに抗凍結剤に対する崩壊速度にきわめて大きな差があった。また、ゲルでコートされた実施例のタンパク質結晶を用いたＸ線回折実験で、ice-ringが生じても、タンパク質結晶自体の損傷は、ほとんど観察されなかった。さらに、シンクロトロン放射光による回折実験でも問題なく十分なデータが得られ、かつ構造解析可能なデータであった。本実施例によれば、リゾチーム、グルコースイソメラーゼ、ソーマチンの３種のタンパク質結晶について、従来のＸ線結晶構造解析結果を上回る最高分解能の反射が得られた。 As described above, according to Examples 1 to 7, all three types of protein crystals (lysozyme, glucose isomerase, thaumatin) obtained in the gel are almost completely prevented from being damaged by the cryoprotectant. It was. That is, when the protein crystal of the example coated with the gel and the free crystal of the comparative example were compared, there was a very large difference in the disintegration rate with respect to the cryoprotectant for all three types of protein crystals. Further, in the X-ray diffraction experiment using the protein crystal of the example coated with the gel, even if ice-ring occurred, the protein crystal itself was hardly damaged. Furthermore, sufficient data was obtained in a diffraction experiment using synchrotron radiation without problems, and the structure analysis was possible. According to the present Example, the highest resolution reflection exceeding the conventional X-ray crystal structure analysis result was obtained for three protein crystals of lysozyme, glucose isomerase, and thaumatin.
［実施例８～１０、比較例８～９］
　ゲルで被覆された被覆結晶を製造し、２．５Ｍ酢酸リチウム水溶液を抗凍結剤として用いてＸ線結晶構造解析を行った。抗凍結剤として２．５Ｍ酢酸リチウム水溶液を用いることと、抗凍結剤への前記被覆結晶の浸漬時間を１５分間とすることと、被解析結晶を構成する生体物質と、ゲル（アガロースＩＩＩ）の濃度以外は、実施例１と同様に行った。前記生体物質として、タンパク質の一種であるエラスターゼを用い、ゲル（アガロースＩＩＩ）の濃度を２．０質量％としたものを実施例８とする。前記生体物質として、タンパク質の一種であるリゾチームを用い、ゲル（アガロースＩＩＩ）の濃度を１．６質量％としたものを実施例９とする。前記生体物質として、タンパク質の一種であるグルコースイソメラーゼを用い、ゲル（アガロースＩＩＩ）の濃度を１．６質量％としたものを実施例１０とする。 [Examples 8 to 10, Comparative Examples 8 to 9]
A coated crystal coated with a gel was produced, and an X-ray crystal structure analysis was performed using a 2.5 M aqueous lithium acetate solution as an antifreezing agent. Using a 2.5M aqueous lithium acetate solution as an antifreezing agent, setting the immersion time of the coated crystal in the antifreezing agent to 15 minutes, the biological material constituting the crystal to be analyzed, and the gel (Agarose III) The same procedure as in Example 1 was performed except for the concentration. Example 8 is one in which elastase, which is a kind of protein, is used as the biological material, and the gel (agarose III) concentration is 2.0 mass%. Example 9 uses lysozyme which is a kind of protein as the biological material, and the concentration of gel (agarose III) is 1.6% by mass. Example 10 uses glucose isomerase, which is a kind of protein, as the biological material and the gel (agarose III) concentration is 1.6 mass%.
　さらに、ゲルを添加しないことと、抗凍結剤（２．５Ｍ酢酸リチウム水溶液）への浸漬時間を変える以外は実施例８および９と同様にして生体物質結晶を製造し、Ｘ線結晶構造解析を行った。前記生体物質として、タンパク質の一種であるエラスターゼを用い、抗凍結剤への浸漬時間を２秒としたものを実施例８とする。前記生体物質として、タンパク質の一種であるリゾチームを用い、抗凍結剤への浸漬時間を１５秒としたものを実施例９とする。 Further, a biological material crystal was produced in the same manner as in Examples 8 and 9 except that the gel was not added and the immersion time in the antifreezing agent (2.5 M lithium acetate aqueous solution) was changed, and the X-ray crystal structure analysis was performed. went. Example 8 is one in which elastase which is a kind of protein is used as the biological material, and the immersion time in the cryoprotectant is 2 seconds. Example 9 uses lysozyme which is a kind of protein as the biological material, and the immersion time in the cryoprotectant is 15 seconds.
　図１１に、実施例８～１０および比較例８～９の結晶の写真およびデータを示す。左から右に向かって、それぞれ、比較例８、実施例８、比較例９、実施例９、および実施例１０の写真である。図下方の数値は、アガロースの添加量（質量％）、抗凍結剤（Ｃｒｙｏ）溶液への浸漬時間、およびＸ線結晶構造解析におけるモザイク性（Ｍｏｓａｉｃｉｔｙ）を示す。図示の通り、実施例８～１０の被覆結晶は、抗凍結剤（２．５Ｍ酢酸リチウム水溶液）に１５分間という長時間浸漬させても結晶が損傷しなかった。これらの結晶のモザイク性は、いずれも約０．３と十分に小さい数値であり、質の高い結晶であることが示された。一方、比較例８の結晶はモザイク性が１．１７と大きく、比較例１０の結晶は、抗凍結剤に１５秒間浸漬させると破損してしまい、Ｘ線結晶構造解析ができなかった。 FIG. 11 shows photographs and data of the crystals of Examples 8 to 10 and Comparative Examples 8 to 9. It is a photograph of comparative example 8, example 8, comparative example 9, example 9, and example 10, respectively from left to right. The numerical value in the lower part of the figure shows the amount of agarose added (mass%), the immersion time in the cryoprotectant (Cryo) solution, and the mosaic property in the X-ray crystal structure analysis. As shown in the figure, the coated crystals of Examples 8 to 10 were not damaged even when immersed for a long time of 15 minutes in an anti-freezing agent (2.5 M lithium acetate aqueous solution). The mosaic property of these crystals is a sufficiently small value of about 0.3, indicating that the crystals are of high quality. On the other hand, the crystal of Comparative Example 8 had a large mosaic property of 1.17, and the crystal of Comparative Example 10 was damaged when immersed in an anti-freezing agent for 15 seconds, and X-ray crystal structure analysis could not be performed.
［実施例１１］
　抗凍結剤（２．５Ｍ酢酸リチウム水溶液）への浸漬時間を１０分間に変える以外は実施例１０と同条件で被覆結晶製造およびＸ線結晶構造解析を行った。その結果、モザイク性の数値は、ビーム分散を含めて０．０８°と、極めて小さい数値であり、従来技術を上回る非常に高品質の結晶であることが示された。図１２に、本実施例の結晶のＸ線回折像を示す。なお、このときの分解能は０．９３Åであり、被解析結晶は、径が０．５ｍｍ以上の大きな結晶であった。 [Example 11]
Coated crystal production and X-ray crystal structure analysis were performed under the same conditions as in Example 10 except that the immersion time in the antifreezing agent (2.5 M lithium acetate aqueous solution) was changed to 10 minutes. As a result, the mosaic value is 0.08 ° including the beam dispersion, which is an extremely small value, indicating that the crystal has a very high quality over the prior art. FIG. 12 shows an X-ray diffraction image of the crystal of this example. The resolution at this time was 0.93 mm, and the crystal to be analyzed was a large crystal having a diameter of 0.5 mm or more.
［実施例１２：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　アガロース－ＩＩＩ溶液（Ａ）におけるアガロース－ＩＩＩ濃度を０～２．０質量％まで、０．２質量％刻みで種々変化させたことと、タンパク質溶液（Ｂ）においてリゾチームの濃度を３０ｍｇ／ｍＬから５０ｍｇ／ｍＬに変えたことと、沈殿化剤溶液における塩化ナトリウム濃度を１．０Ｍから３．０質量％に変えたことと、図４４左下に示したようなバッチ法に適した結晶化プレート（ハンプトンリサーチ社製Ｉｍｐ＠ｃｔ　ｐｌａｔｅ（商品名））を用いてバッチ法にて実施したこと以外は実施例１と同様にして、アガロース－ＩＩＩのゲルで被覆された被覆結晶を製造し、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。なお、以下、図４４左下の図（シッティングドロップ法）の符号を用いて説明するが、この図は模式図であるため、ウェル数、各部の寸法比等は、実際の機器であるＩｍｐ＠ｃｔ　ｐｌａｔｅとは異なる。すなわち、まず、アガロース－ＩＩＩ溶液（Ａ）におけるアガロース－ＩＩＩ濃度を、前述のとおり０～２．０質量％まで０．２質量％刻みで変化させた種々のアガロース－ＩＩＩ濃度で調製した。これと、タンパク質溶液（Ｂ）とを混合させ、アガロース－ＩＩＩ濃度が種々異なる結晶化溶液（Ｃ）を調製した。次に、異なるアガロース－ＩＩＩ濃度を有する前記結晶化溶液（Ｃ）を沈殿化剤溶液と混合した後、６μＬずつ、結晶化プレート２０７のそれぞれのウェルに注入した。さらに、各アガロース－ＩＩＩ濃度について、同じアガロース－ＩＩＩ濃度の結晶化サンプルを合計８つ作製し、それぞれ別個のウェルに注入した。この結晶化溶液（Ｃ）は、ウェル内で液滴を形成し、すぐにゲル化した。その直後、プレート（結晶化プレート）２０７上面にシールを貼って各ウェル中のゲルの大気に対する曝露を防止し、そのまま２０℃で３日間静置したところ、結晶の生成を確認した。その後、各サンプル中に生成した結晶数を顕微鏡で観察した。各濃度における８つのサンプルの生成結晶数を平均し、平均結晶数（個）とした。 [Example 12: Production of crystals under conditions of changing gel concentration and screening of crystallization conditions]
The agarose-III concentration in the agarose-III solution (A) was variously changed from 0 to 2.0% by mass in increments of 0.2% by mass, and the lysozyme concentration in the protein solution (B) was changed from 30 mg / mL. A crystallization plate suitable for the batch method as shown in the lower left of FIG. 44, and the sodium chloride concentration in the precipitating agent solution was changed from 1.0 M to 3.0 mass%. A coated crystal coated with agarose-III gel was produced in the same manner as in Example 1 except that it was carried out by the batch method using Imp @ ct plate (trade name) manufactured by Hampton Research Co., Ltd. The crystallization conditions (crystal production conditions) under the changing conditions were screened. In the following, description will be made using the reference numerals in the lower left of FIG. 44 (sitting drop method). Since this figure is a schematic diagram, the number of wells, the dimensional ratio of each part, etc. It is different from plate. That is, first, the agarose-III concentration in the agarose-III solution (A) was prepared at various agarose-III concentrations changed from 0 to 2.0% by mass in steps of 0.2% by mass as described above. This was mixed with the protein solution (B) to prepare crystallization solutions (C) having different agarose-III concentrations. Next, the crystallization solution (C) having different agarose-III concentrations was mixed with the precipitating agent solution, and 6 μL was injected into each well of the crystallization plate 207. In addition, for each agarose-III concentration, a total of eight crystallized samples with the same agarose-III concentration were prepared and injected into separate wells. This crystallization solution (C) formed droplets in the well and immediately gelled. Immediately thereafter, a seal was affixed to the upper surface of the plate (crystallization plate) 207 to prevent the gel in each well from being exposed to the atmosphere, and left to stand at 20 ° C. for 3 days. Thereafter, the number of crystals formed in each sample was observed with a microscope. The number of produced crystals of eight samples at each concentration was averaged to obtain the average number of crystals (pieces).
　図１５のグラフに、前記平均結晶数を示す。図中、横軸はアガロース－ＩＩＩ濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロース－ＩＩＩ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース－ＩＩＩ存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。 15 shows the average number of crystals. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose-III concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose-III (concentration other than 0), good crystal production conditions can be obtained. It was shown that
［実施例１３：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　アガロースの種類を、アガロース－ＩＩＩ（ＡｇａｒｏｓｅＩＩＩ）、アガロース９Ａ、またはアガロースＳｅａ　Ｐｌａｑｕｅ（アガロースＳＰ）、およびアガロースＳｅａＫｅｍ（いずれも、タカラバイオ株式会社の商品名）としたことと、アガロース濃度を０、０．２、０．４、０．６、０．８、１．０、１．２、１．４、１．６、１．８、２．０、２．５、３．０、３．５、４．０、４．５、５．０、５．５または６．０質量％と種々変化させること（ＳｅａＫｅｍについては２．０質量％以下まで）以外は実施例１２と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。図１６のグラフに、アガロース濃度０～２．０質量％の結果を示す。図左上のグラフは、アガロース９Ａの結果を示すグラフである。図右上のグラフは、アガロースＳＰの結果を示すグラフである。図左下のグラフは、アガロース－ＩＩＩの結果を示すグラフである。図右下のグラフは、アガロースＳｅａＫｅｍの結果を示すグラフである。図中、横軸はアガロース濃度、縦軸は、前記平均結晶数（個）である。また、図１７のグラフに、アガロース濃度０および２．０～６．０質量％の結果を示す。図右上のグラフは、アガロース－ＩＩＩの結果を示すグラフである。図左下のグラフは、アガロース９Ａの結果を示すグラフである。図右下のグラフは、アガロースＳＰの結果を示すグラフである。図中、横軸はアガロース濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロース濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。 [Example 13: Crystal production and crystallization condition screening under gel concentration changing conditions]
The type of agarose was agarose-III (Agarose III), agarose 9A, or agarose Sea Plaque (agarose SP), and agarose SeaKem (all are trade names of Takara Bio Inc.), and the agarose concentration was 0, 0. .2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5 The gel was changed in the same manner as in Example 12 except that it was variously changed to 4.0, 4.5, 5.0, 5.5, or 6.0% by mass (for SeaKem up to 2.0% by mass or less). The crystallization conditions (crystal production conditions) under the concentration change conditions were screened. The graph of FIG. 16 shows the results when the agarose concentration is 0 to 2.0% by mass. The graph on the upper left of the figure is a graph showing the results of agarose 9A. The graph in the upper right of the figure is a graph showing the results of agarose SP. The graph on the lower left of the figure is a graph showing the results of agarose-III. The graph on the lower right of the figure is a graph showing the results of agarose SeaKem. In the figure, the horizontal axis represents the agarose concentration, and the vertical axis represents the average number of crystals (pieces). Further, the graph of FIG. 17 shows the results when the agarose concentration is 0 and 2.0 to 6.0 mass%. The graph in the upper right of the figure is a graph showing the results of agarose-III. The graph on the lower left of the figure is a graph showing the results of agarose 9A. The lower right graph is a graph showing the results of agarose SP. In the figure, the horizontal axis represents the agarose concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (concentration other than 0), it was shown that good crystal production conditions were obtained. It was done.
［実施例１４：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　アガロースの種類を、ＮｕＳｉｅｖｅ３：１（タカラバイオ株式会社の商品名）とすることと、アガロース濃度を０、０．２、０．４、０．６、０．８、１．０、１．２、１．４、１．６、１．８、２．０、２．５、３．０、３．５、４．０、４．５、５．０、５．５または６．０質量％と種々変化させること以外は実施例１２およびと同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。図１８のグラフに、その結果を示す。図中、横軸はＮｕＳｉｅｖｅ３：１濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロースであるＮｕＳｉｅｖｅ３：１濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。また、ＮｕＳｉｅｖｅ３：１の場合、濃度が５．０質量％付近で最も良好な結晶製造条件が得られたことも示された。 [Example 14: Crystal production under crystallization condition change and crystallization condition screening]
The type of agarose is NuSieve 3: 1 (trade name of Takara Bio Inc.), and the agarose concentration is 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 mass% The crystallization conditions (crystal production conditions) under gel concentration change conditions were screened in the same manner as in Example 12 except that various changes were made. The result is shown in the graph of FIG. In the figure, the horizontal axis represents NuSieve 3: 1 concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the correlation mechanism between the agarose NuSieve 3: 1 concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (concentration other than 0), good crystal production conditions were obtained. It was shown that In addition, in the case of NuSieve 3: 1, it was also shown that the best crystal production conditions were obtained when the concentration was around 5.0% by mass.
［実施例１５：ゲル濃度および沈殿化剤濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　沈殿化剤溶液中における塩化ナトリウム濃度を、２．０質量％、３．０質量％、４．０質量％、５．０質量％および６．０質量％と種々変化させることと、沈殿化剤への浸漬時間を６日または７日とすること以外は実施例１２と同様にして、ゲル濃度および沈殿化剤変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。なお、ゲル濃度は、各塩化ナトリウム濃度について、実施例１２と同様、０から２．０質量％まで０．２質量％刻みで変化させた。図１９のグラフに、その結果を示す。図中、横軸はアガロース－ＩＩＩ濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロース－ＩＩＩ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース－ＩＩＩ存在下（濃度が０以外）では、いずれの塩化ナトリウム濃度でも良好な結晶製造条件が得られたことが示された。 [Example 15: Crystal production and crystallization condition screening under changing conditions of gel concentration and precipitant concentration]
Variously changing the concentration of sodium chloride in the precipitant solution to 2.0 mass%, 3.0 mass%, 4.0 mass%, 5.0 mass% and 6.0 mass%; The crystallization conditions (crystal production conditions) under the conditions of changing the gel concentration and the precipitating agent were screened in the same manner as in Example 12 except that the immersion time was 6 days or 7 days. The gel concentration was changed from 0 to 2.0% by mass in increments of 0.2% by mass for each sodium chloride concentration as in Example 12. The result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose-III concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose-III (concentration other than 0), good crystal production is possible at any sodium chloride concentration. It was shown that the conditions were obtained.
［実施例１６：ゲル濃度および沈殿化剤濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　前記各実施例で用いた通常のアガロース－ＩＩＩに代えて高融点アガロース－ＩＩＩを用いること以外は実施例１５と同様にして、ゲル濃度および沈殿化剤変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。なお、ゲル濃度は、各塩化ナトリウム濃度について、実施例１５と同様、０から２．０質量％まで０．２質量％刻みで変化させた。図２０のグラフに、その結果を示す。図中、横軸はアガロース－ＩＩＩ濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロース－ＩＩＩ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース－ＩＩＩ存在下（濃度が０以外）では、いずれの塩化ナトリウム濃度でも良好な結晶製造条件が得られたことが示された。 [Example 16: Crystal production and crystallization condition screening under conditions of changing gel concentration and precipitating agent concentration]
In the same manner as in Example 15 except that high melting point agarose-III was used instead of the normal agarose-III used in each of the above Examples, the crystallization conditions (crystal production under the conditions of changing gel concentration and precipitating agent) Condition). The gel concentration was changed from 0 to 2.0% by mass in increments of 0.2% by mass for each sodium chloride concentration as in Example 15. The result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose-III concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose-III (concentration other than 0), good crystal production is possible at any sodium chloride concentration. It was shown that the conditions were obtained.
［実施例１７：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　アガロース－ＩＩＩ濃度を０、０．４、１．０、１．６または２．０に種々変化させたことと、タンパク質としてリゾチームに代えてエラスターゼを用い、タンパク質溶液（Ｂ）においてエラスターゼの濃度を１２．５ｍｇ／ｍＬとしたことと、アガロースの種類をアガロースＳｅａｐｌａｑｕｅ（ＳＰ）としたことと、平均結晶数の算出に用いるサンプル数を８個に代えて１２個としたこと以外は実施例１２と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。図２１のグラフに、その結果を示す。図中、横軸はアガロースＳＰ濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロースＳＰ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。 [Example 17: Crystal production under crystallization condition change and crystallization condition screening]
Various changes were made to the agarose-III concentration to 0, 0.4, 1.0, 1.6 or 2.0, and elastase was used instead of lysozyme as the protein, and the concentration of elastase in the protein solution (B) was changed. Example 12 except that it was 12.5 mg / mL, that the type of agarose was agarose seaplaque (SP), and that the number of samples used to calculate the average number of crystals was 12 instead of 8. Similarly, crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened. The result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose SP concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated.
［実施例１８：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　タンパク質としてリゾチームに代えてグルコースイソメラーゼを用い、タンパク質溶液（Ｂ）においてグルコースイソメラーゼの濃度を１０ｍｇ／ｍＬとしたことと、アガロースの種類を超低分子量アガロース９Ａとしたことと、沈殿化剤を、塩化ナトリウム水溶液に代えて、１０質量％の２－メチル－２，４－ペンタンジオール（ＭＰＤ）を含む０．１Ｍ塩化カルシウム（ＣａＣｌ２）水溶液としたことと、緩衝液を、０．１Ｍ酢酸バッファーｐＨ４．５に代えて０．１Ｍ　Ｔｒｉｓ－ＨＣｌ　ｐＨ７．０としたこと以外は、実施例１２と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。図２２の写真およびグラフに、その結果を示す。各写真の下方の数値は、アガロース９Ａ濃度（質量％）を示す。グラフ中、横軸はアガロース９Ａ濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロース９Ａ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。 [Example 18: Crystal production under crystallization condition change and crystallization condition screening]
Glucose isomerase was used instead of lysozyme as the protein, the concentration of glucose isomerase was 10 mg / mL in the protein solution (B), the type of agarose was ultra-low molecular weight agarose 9A, and the precipitating agent was chlorinated. Instead of the aqueous sodium solution, a 0.1 M calcium chloride (CaCl 2 ) aqueous solution containing 10% by mass of 2-methyl-2,4-pentanediol (MPD) was used. In the same manner as in Example 12, except that 0.1M Tris-HCl pH 7.0 was used instead of 0.1, crystallization conditions (crystal production conditions) under conditions of changing gel concentration were screened. The results are shown in the photograph and graph of FIG. The numerical value below each photograph indicates the agarose 9A concentration (% by mass). In the graph, the horizontal axis represents the agarose 9A concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose 9A concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (concentration other than 0), good crystal production conditions were obtained in all cases. Indicated.
［実施例１９：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　アガロースの種類をアガロースＳＰとしたことと、沈殿化剤を、８．０質量％の２－メチル－２，４－ペンタンジオール（ＭＰＤ）を含む０．１Ｍ酢酸カルシウム（ＣａＡｃ）水溶液としたこと以外は実施例１８と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。図２３の写真およびグラフに、その結果を示す。各写真の下方の数値は、アガロースＳＰ濃度（質量％）を示す。グラフ中、横軸はアガロースＳＰ濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロースＳＰ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。 [Example 19: Crystal production under crystallization condition change and crystallization condition screening]
Except for the type of agarose being agarose SP and the precipitating agent being a 0.1 M calcium acetate (CaAc) aqueous solution containing 8.0% by mass of 2-methyl-2,4-pentanediol (MPD). In the same manner as in Example 18, crystallization conditions (crystal production conditions) under gel concentration change conditions were screened. The results are shown in the photograph and graph of FIG. The numerical value at the bottom of each photograph indicates the agarose SP concentration (% by mass). In the graph, the horizontal axis represents the agarose SP concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated.
［実施例２０：タンパク質濃度およびゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　タンパク質としてリゾチームに代えてソーマチンを用いたことと、アガロースの種類をＳｅａｐｌａｑｕｅ（タカラバイオ株式会社の商品名）としたことと、沈殿化剤（沈殿剤）を０．３Ｍ　Ｎａ／Ｋ酒石酸塩としたことと、平均結晶数の算出に用いるサンプル数を８個に代えて１２個としたこと以外は実施例１２と同様にして、タンパク質濃度およびゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。図２４のグラフに、その結果を示す。図中、横軸はアガロースＳＰ（Ｓｅａｐｌａｑｕｅ）濃度、縦軸は、前記平均結晶数（個）である。図示の通り、アガロースＳＰ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。また、ソーマチン濃度は、５０ｍｇ／ｍＬでも２５ｍｇ／ｍＬでも、いずれでも良好な結晶製造条件が得られたことが示された。 [Example 20: Crystal production and crystallization condition screening under changing conditions of protein concentration and gel concentration]
The use of thaumatin instead of lysozyme as the protein, the type of agarose as Seaplaque (trade name of Takara Bio Inc.), and the precipitating agent (precipitating agent) as 0.3M Na / K tartrate Except that the number of samples used for calculation of the average number of crystals is 12 instead of 8, and the crystallization conditions (crystal production conditions) under the conditions of changing protein concentration and gel concentration are the same as in Example 12. ) Was screened. The results are shown in the graph of FIG. In the figure, the horizontal axis represents the agarose SP (Seaplaque) concentration, and the vertical axis represents the average number of crystals (pieces). As shown in the figure, the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated. Moreover, it was shown that good crystal production conditions were obtained regardless of whether the thaumatin concentration was 50 mg / mL or 25 mg / mL.
［実施例２１：ゲル濃度変化条件下での結晶製造および結晶化条件スクリーニング］
　下記（１）～（３）以外は実施例１２と同様にして結晶製造および結晶化条件スクリーニングを行った。
（１）ＶＤＸ　ｐｌａｔｅ（ハンプトンリサーチ社の商品名）を用いたハンギングドロップ法により結晶製造を行った。
（２）タンパク質としてリゾチームに代えてＳｙｎｅｃｈｏｃｏｃｃｕｓ由来ホスホリブロキナーゼ（ＰＲＫ）（Ｐｈｏｓｐｈｏｒｉｂｕｌｏｋｉｎａｓｅ（ＰＲＫ）／Ｓｙｎｅｃｈｏｃｏｃｃｕｓ）を用い、これとゲル化剤（アガロース－ＩＩＩに代えてアガロース９Ａを用いた）および４ｍＭのＣＨ２ＨｇＣｌとの混合溶液を結晶化溶液（Ｃ）とした。なお、この結晶化溶液（Ｃ）は、すぐにゲル化するため、調製後、速やかに結晶製造工程に供した。
（３）塩化ナトリウム水溶液および０．１Ｍ酢酸バッファーｐＨ４．５に代えて、１２質量％のイソプロパノール、０．１Ｍまたは０．２２Ｍ酢酸カリウム、および０．１Ｍ　Ｍｅｓ（２－モルホリノエタンスルホン酸）　ｐＨ６．５水溶液を沈殿化剤溶液とした。 [Example 21: Crystal production under crystallization condition change and crystallization condition screening]
Crystal production and crystallization condition screening were performed in the same manner as in Example 12 except for the following (1) to (3).
(1) Crystals were produced by the hanging drop method using VDX plate (trade name of Hampton Research).
(2) Synchococcus-derived phospholibrokinase (PRK) (Phosphoribokinase (PRK) / Synechococcus) is used as a protein instead of lysozyme, and a gelling agent (agarose-III is used instead of agarose-III) and 4 mM CH A mixed solution with 2 HgCl was used as a crystallization solution (C). In addition, since this crystallization solution (C) gelatinized immediately, it used for the crystal manufacturing process immediately after preparation.
(3) 12% by mass of isopropanol, 0.1M or 0.22M potassium acetate, and 0.1M Mes (2-morpholinoethanesulfonic acid) pH 6. 5 aqueous solution was used as the precipitating agent solution.
　ハンギングドロップ法による結晶製造工程は、以下のように行った。すなわち、まず、プレート２０３の各ウェル（プレートウェル）中に、あらかじめ前記沈殿化剤溶液を５００μＬ／ウェル入れて準備した。一方、前記の通り調整した結晶化溶液（Ｃ）を、突起部２０２（カバーガラス）上に２μＬずつ分注してゲル化させた。各濃度の前記結晶化溶液（Ｃ）について、８つの同じサンプルを、８つの突起部２０２の上面に載せた。その後、蓋体２０１を上下反転させて突起部２０２を下に向け、突起部２０２を、前記沈殿化剤溶液を含むプレートウェル中にはめ込み、前記ゲルと前記沈殿化剤溶液とを近接させた。このようにセットした後、２０℃で３日間静置し、ゲル中でＤＮＡ結晶を析出させて結晶を製造した。図２５の写真に、その結果を示す。図中、各写真の下方の数値は、アガロース濃度（質量％）および沈殿化剤（沈殿剤）濃度を示す。図示の通り、アガロース９Ａ濃度（ゲル濃度）と平均結晶数の相関関係のメカニズムは不明であるが、アガロース存在下（濃度が０以外）では、いずれも良好な結晶製造条件が得られたことが示された。 The crystal manufacturing process by the hanging drop method was performed as follows. That is, first, 500 μL / well of the precipitant solution was prepared in advance in each well (plate well) of the plate 203. On the other hand, 2 μL of the crystallization solution (C) prepared as described above was dispensed on the protrusion 202 (cover glass) to be gelled. For each concentration of the crystallization solution (C), eight identical samples were placed on top of the eight protrusions 202. Thereafter, the lid 201 was turned upside down so that the projections 202 faced down, and the projections 202 were fitted into the plate well containing the precipitating agent solution to bring the gel and the precipitating agent solution close to each other. After setting in this way, the mixture was allowed to stand at 20 ° C. for 3 days, and DNA crystals were precipitated in the gel to produce crystals. The result is shown in the photograph of FIG. In the figure, the numerical values below each photograph indicate the agarose concentration (mass%) and the precipitating agent (precipitating agent) concentration. As shown in the figure, the mechanism of the correlation between the agarose 9A concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (concentration other than 0), good crystal production conditions were obtained in all cases. Indicated.
［実施例２２：大腸菌由来タンパク質の結晶製造（結晶化）］
　タンパク質として、前記のタンパク質に代えて大腸菌由来セリンアセチルトランスフェラーゼ（ＳＡＴ）７．５ｍｇ／ｍＬを用いたことと、沈殿化剤（沈殿剤）溶液を４．５質量％のＰＥＧ８０００（Ｐｒｏｍｅｇａ社のポリエチレングリコールの商品名）および０．１Ｍ　カコジル酸ナトリウムを含むｐＨ６．５水溶液としたこと以外は実施例２１と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。 [Example 22: Crystal production of E. coli-derived protein (crystallization)]
As the protein, Escherichia coli-derived serine acetyltransferase (SAT) 7.5 mg / mL was used instead of the above protein, and a precipitating agent (precipitating agent) solution was added to 4.5% by mass of PEG 8000 (Promega's polyethylene glycol) ) And 0.1M sodium cacodylate in pH 6.5 aqueous solution, except that the crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened in the same manner as in Example 21.
［実施例２３：大腸菌由来タンパク質の結晶製造（結晶化）］
　沈殿化剤（沈殿剤）溶液を、７．５質量％のＰＥＧ８０００（Ｐｒｏｍｅｇａ社のポリエチレングリコールの商品名）および０．１Ｍ　カコジル酸ナトリウムを含むｐＨ６．５水溶液としたこと以外は実施例２２と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。 [Example 23: Crystal production of E. coli-derived protein (crystallization)]
Same as Example 22 except that the precipitating agent (precipitating agent) solution was a pH 6.5 aqueous solution containing 7.5% by mass of PEG8000 (trade name of polyethylene glycol from Promega) and 0.1 M sodium cacodylate. Thus, crystallization conditions (crystal production conditions) under gel concentration change conditions were screened.
［実施例２４：大腸菌由来タンパク質の結晶製造（結晶化）］
　タンパク質として、ＳＡＴに代えて大腸菌異物排出トランスポーター（ＡｃｒＢ）２８ｍｇ／ｍＬを用いたことと、沈殿化剤（沈殿剤）溶液を、１０質量％のＰＥＧ２０００（Ｐｒｏｍｅｇａ社のポリエチレングリコールの商品名）、８０ｍＭリン酸二水素ナトリウムおよび２０ｍＭクエン酸ナトリウムを含むｐＨ５．６水溶液としたこと以外は実施例１２と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。 [Example 24: Crystal production of E. coli-derived protein (crystallization)]
As a protein, E. coli foreign body excretion transporter (AcrB) 28 mg / mL was used instead of SAT, and a precipitating agent (precipitating agent) solution was 10% by mass of PEG2000 (trade name of polyethylene glycol of Promega), Crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened in the same manner as in Example 12 except that the aqueous solution was pH 5.6 containing 80 mM sodium dihydrogen phosphate and 20 mM sodium citrate.
［実施例２５：大腸菌由来タンパク質の結晶製造（結晶化）］
　沈殿化剤（沈殿剤）溶液のＰＥＧ２０００濃度を１０質量％に代えて１４質量％としたこと以外は実施例２４と同様にして、ゲル濃度変化条件下での結晶化条件（結晶製造条件）をスクリーニングした。 [Example 25: Crystal production of E. coli-derived protein (crystallization)]
In the same manner as in Example 24 except that the PEG2000 concentration of the precipitating agent (precipitant) solution was changed to 14% by mass instead of 10% by mass, the crystallization conditions (crystal production conditions) under the gel concentration changing conditions were the same. Screened.
　実施例２２～２５の結果、大腸菌由来セリンアセチルトランスフェラーゼ（ＳＡＴ）および大腸菌異物排出トランスポーター（ＡｃｒＢ）のいずれについても、良好な結晶製造（結晶化）条件が得られた。図２６の写真に、実施例２２および２３（ＳＡＴ）についてはアガロース濃度１．０質量％における結果を、実施例２４および２５（ＡｃｒＢ）についてはアガロース濃度２．０質量％における結果を、併せて示す。図左上および中上は、実施例２２の結果を示す写真である。図左下および中下は、実施例２３の結果を示す写真である。図右上は、図２４の結果を示す写真である。図右下は、実施例２５の結果を示す写真である。図示の通り、大腸菌由来のＳＡＴおよびＡｃｒＢのいずれについても、良好な結晶を得ることができた。 As a result of Examples 22 to 25, favorable crystal production (crystallization) conditions were obtained for both the E. coli-derived serine acetyltransferase (SAT) and the E. coli foreign body excretion transporter (AcrB). In the photograph of FIG. 26, the results at an agarose concentration of 1.0% by mass for Examples 22 and 23 (SAT) and the results at an agarose concentration of 2.0% by mass for Examples 24 and 25 (AcrB) are combined. Show. The upper left and middle upper of the figure are photographs showing the results of Example 22. The lower left and middle lower of the figure are photographs showing the results of Example 23. The upper right of the figure is a photograph showing the result of FIG. The lower right of the figure is a photograph showing the results of Example 25. As shown, good crystals could be obtained for both SAT and AcrB derived from E. coli.
［実施例２６：核酸（ＤＮＡ）の結晶製造および結晶化条件スクリーニング］
　以下のようにして、核酸（ＤＮＡ）の結晶を製造した。 [Example 26: Crystal production of nucleic acid (DNA) and screening of crystallization conditions]
A crystal of nucleic acid (DNA) was produced as follows.
　まず、ＤＮＡをアニーリングした。すなわち、まず、一本鎖ＤＮＡであるＤＮＡ１（５’－ＡＡＧＡＡＡＡＡＡＡＡ－３’：配列番号１）の１ｍＭ水溶液と、ＤＮＡ１の相補鎖であるＤＮＡ２（５’－ＴＴＴＴＴＴＴＴＣＴＴ－３’：配列番号２）の１ｍＭ水溶液とを等量混合し、まず６０℃で１０分間、続いて２０℃で１５分間静置した。ここに、ＢＮＡ（Ｂｒｉｄｇｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ、株式会社ジーンデザイン社製）の１ｍＭ水溶液を、前記ＤＮＡ１水溶液と同量加え、２０℃で終夜静置した。このようにして、ＤＮＡのアニーリングを行い、アニール化ＤＮＡ水溶液を得た。このアニール化ＤＮＡ水溶液を、結晶製造に供した。 First, DNA was annealed. That is, first, a 1 mM aqueous solution of DNA1 (5′-AAGAAAAAAA-3 ′: SEQ ID NO: 1), which is a single-stranded DNA, and DNA2 (5′-TTTTTTTCTT-3 ′: SEQ ID NO: 2), which is a complementary strand of DNA1, are used. An equal amount of 1 mM aqueous solution was mixed, and left at 60 ° C. for 10 minutes and then at 20 ° C. for 15 minutes. Here, 1 mM aqueous solution of BNA (Bridged Nucleic Acid, Gene Design Co., Ltd.) was added in the same amount as the DNA1 aqueous solution, and allowed to stand at 20 ° C. overnight. In this way, DNA annealing was performed to obtain an annealed DNA aqueous solution. This annealed DNA aqueous solution was used for crystal production.
　ＶＤＸ　ｐｌａｔｅ（ハンプトンリサーチ社の商品名）を用いたハンギングドロップ法により結晶製造を行うことと、結晶化溶液（Ｃ）として、下記のゲルとＤＮＡとの混合水溶液を用い、沈殿化剤（リザーバー）溶液として下記の溶液を用いること以外は、実施例１２と同様にして結晶製造および結晶化条件スクリーニングを行った。
 
ゲルとＤＮＡとの混合水溶液（濃度は最終濃度）
　　　　　アガロース９Ａ　１．０または１．６質量％
　　　　　１．５質量％　ＭＰＤ　
　　　　　０．５ｍＭ　ＤＮＡ（前記アニール化ＤＮＡ水溶液を左記濃度相当分使用）
　　　　　２０ｍＭ　カコジル酸ナトリウム／１０ｍＭ　ＭｇＣｌ２緩衝液（ｐＨ６．８）
沈殿化剤（リザーバー）溶液
　　　　　１８質量％　ＭＰＤ水溶液（５００μＬ／ウェル）
  Crystal production by the hanging drop method using VDX plate (trade name of Hampton Research), and a precipitating agent (reservoir) using a mixed aqueous solution of gel and DNA described below as the crystallization solution (C) Crystal production and crystallization condition screening were performed in the same manner as in Example 12 except that the following solutions were used as the solution.

Mixed aqueous solution of gel and DNA (concentration is final concentration)
Agarose 9A 1.0 or 1.6% by mass
1.5 mass% MPD
0.5 mM DNA (using the annealed DNA aqueous solution equivalent to the concentration on the left)
20 mM sodium cacodylate / 10 mM MgCl 2 buffer (pH 6.8)
Precipitating agent (reservoir) solution 18% by mass MPD aqueous solution (500 μL / well)
　なお、本実施例では、以下のようにして結晶化溶液（Ｃ）（前記ゲルとＤＮＡとの混合水溶液）を調整した。すなわち、まず、アガロース９Ａを８５℃の水に溶かし、３７℃で保温したゲル化剤水溶液（最終濃度の４倍濃度）を調製した。このゲル化剤水溶液と、ＭＰＤ、カコジル酸ナトリウムおよびＭｇＣｌ２を最終濃度の４倍濃度で含む緩衝液と、前記アニール化ＤＮＡ水溶液とを、この順に１：１：２の体積比で混合し、結晶化溶液（Ｃ）を得た。この結晶化溶液（Ｃ）は、すぐにゲル化するため、調製後速やかに以下の結晶製造工程に供した。 In this example, the crystallization solution (C) (mixed aqueous solution of gel and DNA) was prepared as follows. That is, first, agarose 9A was dissolved in water at 85 ° C., and an aqueous gelling agent solution (4 times the final concentration) kept at 37 ° C. was prepared. This gelling agent aqueous solution, a buffer containing MPD, sodium cacodylate and MgCl 2 at a concentration four times the final concentration, and the annealed DNA aqueous solution are mixed in this order at a volume ratio of 1: 1: 2. A crystallization solution (C) was obtained. Since this crystallization solution (C) was gelated immediately, it was subjected to the following crystal production process immediately after preparation.
　結晶製造工程は、以下のように行った。すなわち、まず、プレート２０３の各ウェル（プレートウェル）中に、あらかじめ前記沈殿化剤溶液を５００μＬ／ウェル入れて準備した。一方、前記の通り調整した結晶化溶液（Ｃ）を、突起部２０２（カバーガラス）上に２μＬずつ分注してゲル化させた。各濃度の前記結晶化溶液（Ｃ）について、８つの同じサンプルを、８つの突起部２０２の上面に載せた。その後、蓋体２０１を上下反転させて突起部２０２を下に向け、突起部２０２を、前記沈殿化剤溶液を含むプレートウェル中にはめ込み、前記ゲルと前記沈殿化剤溶液とを近接させた。このようにセットした後、２０℃で１～５日間静置し、ゲル中でＤＮＡ結晶を析出させて結晶を製造した。 The crystal manufacturing process was performed as follows. That is, first, 500 μL / well of the precipitant solution was prepared in advance in each well (plate well) of the plate 203. On the other hand, 2 μL of the crystallization solution (C) prepared as described above was dispensed on the protrusion 202 (cover glass) to be gelled. For each concentration of the crystallization solution (C), eight identical samples were placed on top of the eight protrusions 202. Thereafter, the lid 201 was turned upside down so that the projections 202 faced down, and the projections 202 were fitted into the plate well containing the precipitating agent solution to bring the gel and the precipitating agent solution close to each other. After setting in this way, the mixture was allowed to stand at 20 ° C. for 1 to 5 days, and DNA crystals were precipitated in the gel to produce crystals.
　図２７の写真に、本実施例におけるＤＮＡ結晶製造（結晶成長）の過程を示す。図２７左欄は、アガロースを加えなかった例（比較例に相当）である。中欄は、アガロース９Ａの最終濃度１．０質量％の例である。右欄は、アガロース９Ａの最終濃度１．６質量％の例である。また、それぞれにおいて、上段は、２０℃で１日静置直後の写真であり、下段は、２０℃で５日静置直後の写真である。図示の通り、アガロースを加えなくてもＤＮＡ結晶は成長するが、アガロースを加えた本発明の実施例によれば、さらに結晶が成長しやすいことが示された。また、図示の通り、２０℃で静置後５日経つと、静置後１日の時よりも、細かい結晶が減ってゲルの透明度が上昇し、結晶が大きく成長した。 FIG. 27 shows a process of producing a DNA crystal (crystal growth) in this example. The left column of FIG. 27 is an example in which agarose was not added (corresponding to a comparative example). The middle column is an example with an agarose 9A final concentration of 1.0 mass%. The right column is an example of a final concentration of 1.6% by mass of agarose 9A. Moreover, in each, the upper stage is a photograph immediately after standing at 20 ° C. for 1 day, and the lower stage is a photograph immediately after standing at 20 ° C. for 5 days. As shown in the figure, the DNA crystal grows without adding agarose, but according to the example of the present invention to which agarose was added, it was shown that the crystal was more likely to grow. Further, as shown in the drawing, when 5 days passed after standing at 20 ° C., the number of fine crystals decreased and the transparency of the gel increased, and the crystals grew larger than when 1 day after standing.
［実施例２７：メビオールジェルを用いた結晶製造および結晶化スクリーニング］
　アガロース－ＩＩＩ溶液（Ａ）に代えて、メビオールジェル濃度を０、１、３および５質量％に種々変化させたメビオールジェル溶液を用いたことと、タンパク質溶液（Ｂ）においてリゾチームの濃度を４０ｍｇ／ｍＬに変えたこと以外は実施例１２と同様にしてリゾチームの結晶を製造し、結晶化スクリーニングを行った。図２８に、その結果を示す。図左側のグラフにおいて、横軸は、メビオールジェル添加量（質量％）であり、縦軸は、平均結晶数（結晶析出数）である。また、右側の写真は、メビオールジェルを１質量％添加した場合およびメビオールジェルを添加しなかった場合において、それぞれ析出した結晶を示す。図示の通り、メビオールジェルを添加しなかった場合は、リゾチーム結晶はほとんど析出せず、析出した結晶も、クラックが発生してしまい、良質な結晶ではなかった。これに対し、メビオールジェルを添加した場合、クラック等のない良質な結晶が多数得られた。特に、メビオールジェル添加量を５質量％に増加させると、結晶析出数が急激に上昇した。さらに、メビオールジェルを７質量％に増加させても、同様に良好な結果が得られた。 [Example 27: Crystal production and crystallization screening using mebiol gel]
Instead of the agarose-III solution (A), a meviol gel solution in which the meviol gel concentration was variously changed to 0, 1, 3, and 5% by mass was used, and the concentration of lysozyme in the protein solution (B) was changed. Lysozyme crystals were produced in the same manner as in Example 12 except that the concentration was changed to 40 mg / mL, and crystallization screening was performed. FIG. 28 shows the result. In the graph on the left side of the figure, the horizontal axis represents the amount of meviol gel added (mass%), and the vertical axis represents the average number of crystals (number of crystal precipitates). Moreover, the photograph on the right side shows the precipitated crystals when 1% by mass of mebiol gel is added and when no mebiol gel is added. As shown in the figure, when no mebiol gel was added, almost no lysozyme crystals were precipitated, and the precipitated crystals were cracked and were not good quality crystals. On the other hand, when mebiol gel was added, many high-quality crystals without cracks were obtained. In particular, when the amount of meviol gel added was increased to 5% by mass, the number of crystal precipitates increased rapidly. Furthermore, even when the mebiol gel was increased to 7% by mass, the same good results were obtained.
　また、上記結晶は、メビオールジェルで被覆された被覆結晶であるが、１５℃以下に冷却することにより、メビオールジェルが液化（ゾル化）し、極めて簡便にゲル被覆を除去することができた。図２９の写真に、その様子を示す。左側の写真は、冷却前の、メビオールジェルで被覆された結晶を示す。右側の写真は、冷却後の、ゲル被覆が除去された結晶を示す。 The above crystals are coated crystals coated with mebiol gel. By cooling to 15 ° C. or lower, mebiol gel is liquefied (solified) and the gel coating can be removed very easily. It was. This is shown in the photograph of FIG. The photograph on the left shows crystals coated with mebiol gel before cooling. The photo on the right shows the crystal after cooling, with the gel coating removed.
［ゲル被覆結晶の耐乾燥性］
　実施例１３および２７における結晶化スクリーニングで製造した被覆結晶の対乾燥性を評価した。実施例１３は、２．０質量％アガロース９Ａ、２．０質量％アガロースＳＰ、または２．０質量％アガロース－ＩＩＩを用いて製造した被覆結晶をそれぞれ評価に用いた。実施例２７は、メビオールジェル１質量％を用いて製造した被覆結晶を評価に用いた。評価は、結晶の入った前記ハンギングドロップ法用の装置（ハンプトンリサーチ社製ＶＤＸ　ｐｌａｔｅ（商品名））ウェル上面からシールを取り除いて結晶を常温の大気に曝露し、経時変化を顕微鏡観察することで行った。 [Drying resistance of gel-coated crystals]
The dryness of the coated crystals produced by the crystallization screening in Examples 13 and 27 was evaluated. In Example 13, coated crystals produced using 2.0 mass% agarose 9A, 2.0 mass% agarose SP, or 2.0 mass% agarose-III were used for evaluation. In Example 27, a coated crystal produced using 1% by mass of meviol gel was used for evaluation. The evaluation is performed by removing the seal from the upper surface of the well (VDX plate (trade name) manufactured by Hampton Research Co., Ltd.) for the hanging drop method containing the crystal, exposing the crystal to normal temperature atmosphere, and observing the change with time under a microscope. went.
　図３０の写真に、実施例１３の結晶の対乾燥性評価結果を示す。１番左の「Ｎｏｎｅ」のラインは、ゲルを添加しないで製造した結晶（比較例）の経時変化を示す。左から２番目の「２．０％　９Ａ」のラインは、２．０質量％アガロース９Ａを用いて製造した結晶の経時変化を示す。左から３番目の「２．０％　ＳＰ」のラインは、２．０質量％アガロースＳＰを用いて製造した結晶の経時変化を示す。一番右の「２．０％　ＩＩＩ」のラインは、２．０質量％アガロース－ＩＩＩを用いて製造した結晶の経時変化を示す。写真の左側における「ｍｉｎ」で表した数字は、結晶の、大気に対する曝露時間（分）を表す。図示の通り、ゲルを添加しないで製造した結晶（比較例）は、曝露時間１０分ですでに乾燥が始まり、曝露時間１６分では顕著なクラックが起こり、すでに実用に耐えず、曝露時間２９分では完全に崩壊していた。これに対し、実施例の各被覆結晶は、曝露時間２０～２９分という長時間、乾燥に耐えた。 30 shows the results of evaluation of the crystal of Example 13 against dryness. The leftmost “None” line shows the change with time of a crystal (comparative example) produced without adding gel. The second “2.0% 9A” line from the left shows the change over time of crystals produced using 2.0 mass% agarose 9A. The third “2.0% SP” line from the left shows the change over time of crystals produced using 2.0 mass% agarose SP. The rightmost “2.0% III” line shows the time course of the crystals produced using 2.0 mass% agarose-III. The number represented by “min” on the left side of the photograph represents the exposure time (minutes) of the crystal to the atmosphere. As shown in the figure, the crystal produced without the addition of the gel (comparative example) started to dry at an exposure time of 10 minutes, a remarkable crack occurred at an exposure time of 16 minutes, and was already unusable, and the exposure time was 29 minutes. Then it was completely collapsed. On the other hand, each coated crystal of the example withstood the drying for a long time of exposure time of 20 to 29 minutes.
　さらに、図３１Ａおよび３１Ｂの写真に、実施例２７の結晶の対乾燥性評価結果を示す。図３１Ａ上段の「ゲル添加なし」のラインは、ゲルを添加しないで製造した結晶（比較例）の経時変化を示す。図３１Ａ下段の「メビオール１％添加」のラインは、１．０質量％メビオールジェルを用いて製造した結晶の経時変化を示す。図３１Ｂ上段は、図３１Ａ上段に引き続き、ゲルを添加しないで製造した結晶（比較例）の経時変化を示す。図３１Ｂ下段は、図３１Ａ下段に引き続き、１．０質量％メビオールジェルを用いて製造した結晶の経時変化を示す。「ｍｉｎ」で表した数字は、結晶の、大気に対する曝露時間（分）を表す。図示の通り、ゲルを添加しないで製造した結晶（比較例）は、曝露時間４分ですでに表面の乾燥がはじまり、曝露時間２２分でクラックが入り始め、曝露時間２８分では顕著な沈殿やひび割れが生じ、曝露時間３６分でほぼ完全に崩壊した。これに対し、メビオールジェルで被覆された実施例の被覆結晶は、曝露時間３６分でもクラックや乾燥等の劣化を何ら起こさず、曝露時間４１分でも、ごく一部の結晶に微細なクラックが入ったのみであった。 Furthermore, the photographs of FIGS. 31A and 31B show the results of evaluation of the crystal of Example 27 against dryness. The “no gel addition” line in the upper part of FIG. 31A shows the change with time of the crystal (comparative example) produced without adding the gel. The line of “addition of 1% mebiol” in the lower part of FIG. 31A shows the change over time of crystals produced using 1.0 mass% meviol gel. The upper part of FIG. 31B shows the change with time of the crystal (comparative example) produced without adding the gel, following the upper part of FIG. 31A. The lower part of FIG. 31B shows the change over time of crystals produced using 1.0 mass% meviol gel following the lower part of FIG. 31A. The number represented by “min” represents the exposure time (minutes) of the crystal to the atmosphere. As shown in the figure, the crystal produced without the addition of gel (comparative example) started to dry on the surface after exposure time of 4 minutes, began to crack after exposure time of 22 minutes, Cracks occurred and almost completely collapsed after 36 minutes of exposure. On the other hand, the coated crystal of the example coated with mebiol gel does not cause any deterioration such as cracking and drying even at an exposure time of 36 minutes, and even a small amount of crystals have fine cracks even at an exposure time of 41 minutes. Only entered.
　このように、本発明の製造方法により製造される被覆結晶は、ゲルで被覆されていることにより、乾燥に極めて強く、保存に向くことが確認された。 Thus, it was confirmed that the coated crystal produced by the production method of the present invention was extremely resistant to drying and suitable for storage because it was coated with a gel.
［実施例２８：メビオールジェルを用いた結晶製造］
　リゾチーム濃度　３５ｍｇ／ｍＬ、塩化ナトリウム濃度　３質量％、酢酸ナトリウム濃度を０．２Ｍ、メビオールジェル濃度　２質量％の混合溶液を１２℃に保ち、そのまま１２℃で約１週間静置して結晶を析出させ、本発明の結晶製造方法を実施した。さらにその後、前記混合溶液の温度を２０℃に上昇し、ゲル状態に変換（移動）させた。この状態変化（移動）の前後で結晶に損傷等の変化があるか観察した。図３２の写真に、その観察結果を示す。左はゾル状態（移動前）の写真であり、右はゲル状態（移動後）の写真である。図示の通り、移動前後で結晶の状態に変化は見られなかった。さらに、メビオールジェル濃度を３質量％に代えても同様の結果が得られた。 [Example 28: Crystal production using mebiol gel]
A mixed solution of lysozyme concentration 35 mg / mL, sodium chloride concentration 3% by mass, sodium acetate concentration 0.2M, mebiol gel concentration 2% by mass is kept at 12 ° C. and allowed to stand at 12 ° C. for about 1 week. The crystal production method of the present invention was carried out. Thereafter, the temperature of the mixed solution was raised to 20 ° C. and converted (moved) into a gel state. It was observed whether there was a change such as damage to the crystal before and after this state change (movement). The observation result is shown in the photograph of FIG. The left is a photograph of the sol state (before movement), and the right is a photograph of the gel state (after movement). As shown, no change was observed in the crystal state before and after the movement. Further, similar results were obtained even when the meviol gel concentration was changed to 3% by mass.
　図３３の表および写真に、実施例２７および２８の結晶のＸ線結晶構造解析の結果を示す。Ｘ線結晶構造解析は、前述の各実施例と同様に行った。抗凍結剤は２．５Ｍ酢酸リチウムを用い、浸漬時間は１５分間とした。表中の番号１は、メビオールジェルを添加せずに製造した結晶（比較例）を示す。番号２および３は、実施例２８の結晶（メビオールジェル２質量％および３質量％）を示す。番号４および５は、実施例２７の結晶（メビオールジェル２質量％および５質量％）を示す。写真は、Ｘ線回折像を示し、写真に付された１、２、３および５の番号は、それぞれ表中の番号に対応する。図示のとおり、いずれの結晶においても良好な解析結果が得られ、メビオールジェルの添加および結晶析出後のゲル化は、結晶に対し何ら悪影響を及ぼさないことが確認された。なお、番号２の結晶のみ、若干モザイク性の数値が大きくなっているが、これは、結晶を前記ＶＤＸ　ｐｌａｔｅから剥がす際の操作エラーにより、若干損傷が起こってしまったためであり、結晶構造の本質的な欠陥によるものではない。 33 shows the results of X-ray crystal structure analysis of the crystals of Examples 27 and 28 in the table and photograph of FIG. X-ray crystal structure analysis was carried out in the same manner as in the previous examples. The cryoprotectant was 2.5M lithium acetate and the immersion time was 15 minutes. Number 1 in the table indicates a crystal (comparative example) produced without adding mebiol gel. Numbers 2 and 3 show the crystals of Example 28 (2% and 3% by weight meviol gel). Numbers 4 and 5 indicate the crystals of Example 27 (2% and 5% by weight meviol gel). The photograph shows an X-ray diffraction image, and the numbers 1, 2, 3, and 5 attached to the photograph respectively correspond to the numbers in the table. As shown in the figure, good analysis results were obtained for any crystal, and it was confirmed that the addition of meviol gel and gelation after crystal precipitation had no adverse effect on the crystal. Note that only the number 2 crystal has a slightly larger mosaic value, because this was caused by a slight damage due to an operation error when peeling the crystal from the VDX plate. It is not due to a flaw.
　さらに、実施例２７のリゾチーム結晶のうち、メビオールジェルを７質量％添加して製造したものについて、同様にＸ線結晶構造解析を行った。図３４に、その結果を示す。図３４左側に示すように、良好な結晶が得られ、右側に示すように、良好なＸ線回折像と低いモザイク性（０．３１７８）が得られた。 Further, among the lysozyme crystals of Example 27, those produced by adding 7% by mass of meviol gel were subjected to X-ray crystal structure analysis in the same manner. FIG. 34 shows the result. As shown on the left side of FIG. 34, a good crystal was obtained, and as shown on the right side, a good X-ray diffraction image and a low mosaic property (0.3178) were obtained.
［フェムト秒レーザーによる被覆結晶の加工による加工結晶の製造］
　実施例１８で製造したグルコースイソメラーゼのアガロースＳＰゲル被覆結晶（アガロースＳＰ濃度１．６質量％）をフェムト秒レーザーで加工し、一部を切り出し、加工結晶を製造した。レーザ光源としては、サイバーレーザー社製イフリート（商品名）を用い、出力は０．７～２．０ｍＷとした。図３５の写真に、その様子を示す。図左は加工前であり、図右は加工後である。図示のように、フェムト秒レーザーで加工した加工部において、結晶およびゲルの一部を切り出し、加工結晶を製造することができた。 [Production of processed crystal by processing of coated crystal with femtosecond laser]
The agarose SP gel-coated crystal of glucose isomerase produced in Example 18 (agarose SP concentration 1.6% by mass) was processed with a femtosecond laser, and a part thereof was cut out to produce a processed crystal. As the laser light source, Efreet (trade name) manufactured by Cyber Laser Co., Ltd. was used, and the output was 0.7 to 2.0 mW. This is shown in the photograph of FIG. The left side of the figure is before processing, and the right side of the figure is after processing. As shown in the figure, in the processed part processed by the femtosecond laser, a part of the crystal and the gel was cut out, and the processed crystal could be manufactured.
　加工により切り出した加工部（加工結晶）は、ピンセットやクライオループなどを用いて取り出し、Ｘ線結晶構造解析に供することができた。図３６左は、前記加工部（加工結晶）を取り出している最中の状態を示す写真であり、図３６右は、取り出し後の前記加工部（加工結晶）の写真である。また、図３７に、前記加工部のＸ線結晶構造解析結果を示す。図３７左は、前記加工部の結晶とゲルを示す写真であり、図右は、図左に示した各部分の分解能およびモザイク性である。図示のように、加工部に含まれる結晶は、良好な構造を有することが確認された。 The processed part (processed crystal) cut out by processing could be taken out using tweezers, cryoloop, etc., and used for X-ray crystal structure analysis. The left side of FIG. 36 is a photograph showing a state in which the processed part (processed crystal) is being taken out, and the right part of FIG. 36 is a photograph of the processed part (processed crystal) after being taken out. FIG. 37 shows the X-ray crystal structure analysis result of the processed part. The left side of FIG. 37 is a photograph showing the crystal and gel of the processed part, and the right side is the resolution and mosaic property of each part shown on the left side of the figure. As shown in the figure, it was confirmed that the crystal included in the processed part has a good structure.
　このように、フェムト秒レーザー等のレーザー光を用いた加工によれば、例えば、ゲル被覆結晶から、ゲルおよび結晶のうち使用目的に供する部分のみを適切に切り出すことができる。また、例えば、結晶からゲルのみを適切に除去し、前記ゲルを含まない結晶だけを取り出すこともできる。 Thus, according to processing using a laser beam such as a femtosecond laser, for example, it is possible to appropriately cut out only the portion of the gel and crystal that serves the purpose of use from the gel-coated crystal. Further, for example, it is possible to appropriately remove only the gel from the crystal and take out only the crystal not containing the gel.
　このようなレーザー光加工は、ゲルで被覆されていない結晶に対しても行うことができる。このレーザー光加工によれば、密封環境下で非接触加工を行うことができる。さらに、光エネルギーで分子結合を切断するため、周辺部分に熱拡散せずに加工を行うことができる。特に、フェムト秒レーザーは、集光点付近の例えば数μｍの部分でのみ光エネルギー吸収が起こるため、精密な三次元加工等が可能である。しかしながら、液中に存在しゲルで被覆されていない結晶をレーザー光で加工した場合、結晶がゲルで固定されておらず動いてしまうために、正確な加工がしにくい場合がある。また、結晶がゲルで保護されていないために損傷してしまうおそれがある。さらに、加工の際のデブリ（切りくずや結晶の破片など）が拡散して結晶表面に再付着し、多結晶化等を引き起こしてしまうおそれもある。しかし、レーザー光加工に用いる結晶が、ゲルで被覆された被覆結晶（ゲル被覆結晶）であれば、これらの問題を解決することができる。一方、ゲル被覆結晶にレーザー光を用いて加工すれば、物理的手段による加工よりも、結晶の加工や取出しを簡便に行うことができ、ゲル被覆が加工の妨げになりにくいという利点がある。このように、ゲル被覆結晶のレーザー光加工であれば、ゲル被覆結晶とレーザー光加工との双方の課題を補うことができるのである。 Such laser beam processing can be performed on crystals not covered with gel. According to this laser beam processing, non-contact processing can be performed in a sealed environment. Furthermore, since the molecular bond is cut by light energy, processing can be performed without thermal diffusion to the peripheral portion. In particular, the femtosecond laser absorbs light energy only in the vicinity of, for example, a few μm near the condensing point, so that precise three-dimensional processing or the like is possible. However, when a crystal that is present in the liquid and is not coated with a gel is processed with a laser beam, the crystal is not fixed with the gel and moves, so that accurate processing may be difficult. Moreover, since the crystal is not protected by the gel, it may be damaged. Furthermore, debris (such as chips and crystal fragments) at the time of processing may diffuse and reattach to the crystal surface, causing polycrystallization. However, if the crystal used for laser beam processing is a coated crystal coated with a gel (gel-coated crystal), these problems can be solved. On the other hand, if the gel-coated crystal is processed using laser light, the crystal can be processed and taken out more easily than the processing by physical means, and the gel coating has an advantage that the processing is not hindered. Thus, if it is laser beam processing of a gel coat crystal, the subject of both gel coat crystal and laser beam processing can be supplemented.
［実施例２９：被覆結晶を種結晶として用いた成長結晶製造］
　実施例１２の結晶化スクリーニングにおいて製造した、アガロースゲルで被覆されたリゾチーム結晶（アガロース－ＩＩＩ濃度１．０質量％）の一部を、前述のフェムト秒レーザーにより切り出して種結晶とした。この種結晶を、塩化ナトリウム３質量％およびリゾチーム２０ｍｇ／ｍＬを含む水溶液中に入れ、室温で６０日間静置して結晶を成長させ、本発明の成長結晶製造方法を実施した。図３８の写真に、その様子を示す。左図は、実施例１２のゲル被覆結晶であり、中図は、その一部をフェムト秒レーザー加工で切り出した種結晶（加工結晶）であり、右図は、室温で６０日間成長させた後の成長結晶である。図示の通り、溶液中での成長により、種結晶よりもはるかに大きな、径が約１ｍｍにも達する大きな結晶が得られた。このような大きな結晶は、種々の用途に用いることができ、例えば、中性子線結晶構造解析等に向く。 [Example 29: Production of growth crystal using coated crystal as seed crystal]
A part of the lysozyme crystal (agarose-III concentration: 1.0% by mass) coated with the agarose gel produced in the crystallization screening of Example 12 was cut out with the above-mentioned femtosecond laser as a seed crystal. This seed crystal was put in an aqueous solution containing 3% by mass of sodium chloride and 20 mg / mL of lysozyme, and allowed to stand at room temperature for 60 days to grow the crystal, and the growth crystal production method of the present invention was carried out. This is shown in the photograph of FIG. The left figure is the gel-coated crystal of Example 12, the middle figure is a seed crystal (processed crystal) that is partly cut by femtosecond laser processing, and the right figure is after growth for 60 days at room temperature. It is a grown crystal. As shown in the figure, growth in a solution yielded a large crystal that was much larger than the seed crystal and had a diameter of about 1 mm. Such a large crystal can be used for various applications, and is suitable for, for example, neutron beam crystal structure analysis.
［実施例３０：被覆結晶を種結晶として用いた成長結晶製造］
　実施例２７の結晶化スクリーニングにおいて製造した、メビオールジェルで被覆されたリゾチーム結晶（メビオールジェル濃度５．０質量％）の一部を、前述のフェムト秒レーザーにより切り出して種結晶とした。この種結晶を、塩化ナトリウム３質量％およびリゾチーム２０ｍｇ／ｍＬを含む水溶液中に入れ、室温で４日間静置して結晶を成長させ、本発明の成長結晶製造方法を実施した。図３９の写真に、その様子を示す。左図は、実施例２７のゲル被覆結晶であり、中図は、その一部をフェムト秒レーザー加工で切り出した種結晶（加工結晶）であり、右図は、室温で４日間成長させた後の成長結晶である。図示の通り、溶液中での成長により、種結晶よりもはるかに大きな、径が約０．５ｍｍにも達する大きな結晶が得られた。このような大きな結晶は、実施例２９でも述べた通り、種々の用途に用いることができ、例えば、中性子線結晶構造解析等に向く。 [Example 30: Production of growth crystal using coated crystal as seed crystal]
A part of the lysozyme crystal coated with meviol gel (meviol gel concentration: 5.0% by mass) produced in the crystallization screening of Example 27 was cut out with the femtosecond laser as a seed crystal. This seed crystal was placed in an aqueous solution containing 3% by mass of sodium chloride and 20 mg / mL of lysozyme, and allowed to stand at room temperature for 4 days to grow a crystal, and the growth crystal production method of the present invention was carried out. This is shown in the photograph of FIG. The left figure is the gel-coated crystal of Example 27, the middle figure is a seed crystal (processed crystal) that is partly cut by femtosecond laser processing, and the right figure is after growth for 4 days at room temperature. It is a grown crystal. As shown in the figure, growth in a solution yielded a large crystal that was much larger than the seed crystal and had a diameter of about 0.5 mm. Such a large crystal can be used for various applications as described in Example 29, and is suitable for, for example, neutron beam crystal structure analysis.
［実施例３１：遠心分離による濃度勾配を用いた結晶化スクリーニング］
　遠心分離機を用い、遠心分離チューブ内部の上部と下部とで沈殿剤（沈殿化剤）濃度に勾配を形成することで、結晶化スクリーニングを行った。以下、図４０を用いて具体的に説明する。 [Example 31: Crystallization screening using concentration gradient by centrifugation]
Crystallization screening was performed by forming a gradient in the concentration of the precipitating agent (precipitating agent) between the upper and lower parts inside the centrifuge tube using a centrifuge. Hereinafter, this will be specifically described with reference to FIG.
　すなわち、まず、遠心分離チューブ４０５（ＢＤ社製、商品名　ＢＤ　Ｆａｌｃｏｎ　Ｃｅｌｌ　Ｃｕｌｔｕｒｅ　Ｉｎｓｅｒｔｓ）を準備した。この遠心分離チューブは、底部にＰＥＴ製の半透膜（透析膜）が貼られており、前記半透膜は、固形物は通さずに、一定の大きさ以下の粒子のみを通す。次に、この遠心分離チューブ４０５内部に、２．０質量％アガロース水溶液（アガロースの種類は、Ｓｅａｐｌａｑｕｅ）を６５０μＬ入れ、ゲル化させた。さらにそのゲルの上面に、リゾチーム５０ｍｇ／ｍＬ、塩化ナトリウム４．０質量％、０．１Ｍ　酢酸ナトリウム　ｐＨ４．５を含む水溶液６５０μＬを注いだ。これを、遠心分離機器（久保田商事株式会社社製、商品名　Ｐｌａｔｅ　Ｓｐｉｎ）を用いて、２０℃において２０，０００ｒｐｍで１５時間遠心分離し、前記ゲル中にリゾチーム溶液を均一に拡散させた。 That is, first, a centrifuge tube 405 (manufactured by BD, trade name: BD Falcon Cell Culture Inserts) was prepared. This centrifuge tube has a PET semipermeable membrane (dialysis membrane) attached to the bottom, and the semipermeable membrane allows only particles of a certain size or less to pass through without passing through solids. Next, 650 μL of a 2.0 mass% agarose aqueous solution (agarose type is Seaplaque) was placed in the centrifuge tube 405 and gelled. Further, 650 μL of an aqueous solution containing 50 mg / mL of lysozyme, 4.0% by mass of sodium chloride, 0.1M sodium acetate, pH 4.5 was poured onto the upper surface of the gel. This was centrifuged at 20,000 rpm for 15 hours at 20 ° C. using a centrifuge (trade name Plate Spin, manufactured by Kubota Corporation), and the lysozyme solution was uniformly diffused in the gel.
　一方、遠心分離チューブ４０５よりも太く底部が閉じられた円筒容器４０６中に、沈殿化剤（４．０質量％塩化ナトリウム水溶液）１０００μＬを入れた。さらにその中に、前記ゲルを含んだ遠心分離チューブ４０５を入れた。そのまま２０℃で２日間静置すると、遠心分離チューブ４０５内部には、底部のＰＥＴ膜を通じて徐々に沈殿化剤が浸透し、下部では沈殿化剤濃度が高く、上部では沈殿化剤濃度が低くなり、前記ゲル中にリゾチーム結晶が析出した。このようにして、前記ゲル中で結晶を析出させてリゾチーム結晶を製造した。 Meanwhile, 1000 μL of a precipitating agent (4.0 mass% sodium chloride aqueous solution) was placed in a cylindrical container 406 that was thicker than the centrifuge tube 405 and closed at the bottom. Further, a centrifuge tube 405 containing the gel was put therein. When left at 20 ° C. for 2 days, the precipitating agent gradually permeates into the centrifuge tube 405 through the bottom PET film, and the concentration of the precipitating agent is high at the bottom and the concentration of the precipitating agent is low at the top. A lysozyme crystal was precipitated in the gel. In this way, crystals were precipitated in the gel to produce lysozyme crystals.
　結晶製造（析出）後の遠心分離チューブ４０５内におけるゲル内部を、実体顕微鏡で観察した。撮影は、遠心分離チューブ４０５（カップ）内部の下部（ＰＥＴ膜（メンブレン）側）から上部にかけて、複数の異なる角度から行った。図４１に、その写真を示す。図示の通り、図４１上部は、遠心分離チューブ４０５（カップ）内部の上部を示し、図４１下部は、遠心分離チューブ４０５（カップ）内部の下部（ＰＥＴ膜（メンブレン）側）を示す。同図から分かる通り、沈殿化剤濃度が高いカップ下部（メンブレン側）では析出した結晶の個数が多かった。逆に、沈殿化剤濃度が低いカップ上部では、析出した結晶の個数が少なかった。すなわち、遠心分離を用いて、遠心分離容器内部の上部と下部とで沈殿化剤の濃度勾配を形成し、結晶化条件のスクリーニングを行うことができた。なお、この実施例は、沈殿化剤の濃度勾配を利用した結晶化条件のスクリーニングであるが、同様にタンパク質濃度勾配を形成することによる結晶化条件のスクリーニングも簡便に行うことができる。タンパク質以外の生体物質に対しても同様である。 The inside of the gel in the centrifuge tube 405 after crystal production (precipitation) was observed with a stereomicroscope. Photographing was performed from a plurality of different angles from the lower part (PET film (membrane) side) inside the centrifuge tube 405 (cup) to the upper part. FIG. 41 shows the photograph. 41, the upper part of FIG. 41 shows the upper part inside the centrifuge tube 405 (cup), and the lower part of FIG. 41 shows the lower part inside the centrifuge tube 405 (cup) (PET membrane (membrane) side). As can be seen from the figure, the number of precipitated crystals was large at the lower part of the cup (membrane side) where the precipitant concentration was high. On the contrary, in the upper part of the cup having a low precipitating agent concentration, the number of precipitated crystals was small. That is, using centrifuging, a concentration gradient of the precipitating agent was formed between the upper part and the lower part inside the centrifuge container, and the crystallization conditions could be screened. In addition, although this Example is a screening of the crystallization conditions using the concentration gradient of a precipitating agent, the screening of the crystallization conditions by forming a protein concentration gradient can also be performed simply. The same applies to biological substances other than proteins.
　以上の通り、本発明の結晶製造方法によれば、溶液中から結晶を析出させるよりも、前記生体物質の結晶を簡便に製造することが可能である。また、本発明の凍結結晶製造方法によれば、前記結晶が凍結による損傷を受けにくい。さらに、本発明の成長結晶製造方法によれば、前記ゲルで被覆された被覆結晶を種結晶として結晶を成長させるため、より大きな結晶を得ることができる。また、本発明の結晶は、前記本発明の結晶製造方法、前記本発明の凍結結晶製造方法、または前記本発明の成長結晶製造方法により製造されることで、例えば結晶構造解析等に向いた良好な特性を有する。さらに、本発明の構造解析方法によれば、前記ゲルで被覆された被覆結晶またはそれを凍結した前記凍結結晶を構造解析するため、結晶の取り扱いがしやすいという利点がある。 As described above, according to the crystal manufacturing method of the present invention, it is possible to easily manufacture a crystal of the biological substance, rather than precipitating the crystal from a solution. Moreover, according to the frozen crystal manufacturing method of the present invention, the crystal is not easily damaged by freezing. Furthermore, according to the growth crystal manufacturing method of the present invention, since the crystal is grown using the coated crystal coated with the gel as a seed crystal, a larger crystal can be obtained. In addition, the crystal of the present invention is manufactured by the crystal manufacturing method of the present invention, the frozen crystal manufacturing method of the present invention, or the grown crystal manufacturing method of the present invention, so that it is suitable for crystal structure analysis, for example. It has special characteristics. Furthermore, according to the structure analysis method of the present invention, since the structure analysis of the coated crystal coated with the gel or the frozen crystal frozen from the gel is performed, there is an advantage that the crystal is easy to handle.
　さらに、本発明の結晶化スクリーニング方法は、本発明の結晶製造方法により簡便に結晶を製造できることで、結晶製造条件をさほど厳密に設定しなくても良いため、スクリーニングをも簡便に行うことができる。さらに、本発明の結晶化スクリーニング装置は、同様の理由により、装置の構成を簡略化することが可能である。 Furthermore, since the crystallization screening method of the present invention can easily produce crystals by the crystal production method of the present invention, it is not necessary to set the crystal production conditions so strictly, and thus screening can be performed easily. . Furthermore, the crystallization screening apparatus of the present invention can simplify the configuration of the apparatus for the same reason.
　本発明の結晶製造方法および凍結結晶製造方法は、結晶構造解析、特にＸ線結晶構造解析に有用であり、さらに、結晶化スクリーニング方法にも応用可能である。さらに、本発明の結晶製造方法および凍結結晶製造方法と、それらにより製造された結晶および凍結結晶の用途は、前記の用途に限定されず、あらゆる用途に使用可能である。 The crystal production method and frozen crystal production method of the present invention are useful for crystal structure analysis, particularly X-ray crystal structure analysis, and can also be applied to crystallization screening methods. Furthermore, the method for producing crystals and the method for producing frozen crystals of the present invention and the uses of the crystals and frozen crystals produced thereby are not limited to the above-mentioned uses, and can be used for all uses.

Claims (16)

  1. 　生体物質を結晶化させる結晶化工程を含む、前記生体物質の結晶の製造方法であって、
    　前記結晶化工程において、前記生体物質をゲル中で結晶化させることを特徴とする製造方法。     A method for producing a crystal of the biological material, comprising a crystallization step of crystallizing the biological material,
    In the crystallization step, the biological material is crystallized in a gel.
  2. 　前記生体物質が、生体高分子化合物、タンパク質、天然タンパク質、人工タンパク質、ペプチド、天然ペプチド、人工ペプチド、核酸、天然核酸、人工核酸、糖鎖、天然糖鎖、または人工糖鎖である請求項１記載の製造方法。 2. The biological material is a biopolymer compound, protein, natural protein, artificial protein, peptide, natural peptide, artificial peptide, nucleic acid, natural nucleic acid, artificial nucleic acid, sugar chain, natural sugar chain, or artificial sugar chain. The manufacturing method as described.
  3. 　前記結晶化工程に先立ち、
    　前記生体物質の溶液を準備する溶液準備工程と、
    　前記溶液をゲル化させて前記ゲルを調製するゲル化工程とをさらに含む、
    　請求項１の製造方法。     Prior to the crystallization step,
    A solution preparation step of preparing a solution of the biological material;
    Further comprising a gelling step of preparing the gel by gelling the solution,
    The manufacturing method of Claim 1.
  4. 　前記生体物質溶液がゲル化剤をさらに含む請求項３記載の製造方法。 The method according to claim 3, wherein the biological material solution further contains a gelling agent.
  5. 　ゲル化剤が、多糖類、増粘多糖類、タンパク質、および昇温時ゲル化型ゲルからなる群から選択される少なくとも一つである請求項４記載の製造方法。 The production method according to claim 4, wherein the gelling agent is at least one selected from the group consisting of polysaccharides, thickening polysaccharides, proteins, and gels at elevated temperature.
  6. 　ゲル化剤が、アガロース、寒天、カラギーナン、ゼラチン、コラーゲン、ポリアクリルアミド、昇温時ゲル化型ポリアクリルアミドゲルからなる群から選択される少なくとも一つである請求項４記載の製造方法。 The method according to claim 4, wherein the gelling agent is at least one selected from the group consisting of agarose, agar, carrageenan, gelatin, collagen, polyacrylamide, and gelled polyacrylamide gel at elevated temperature.
  7. 　前記ゲルのゲル強度が１００Ｐａ以上である請求項１記載の製造方法。 The method according to claim 1, wherein the gel has a gel strength of 100 Pa or more.
  8. 　製造される前記結晶が、前記ゲルで被覆された被覆結晶である請求項１記載の製造方法。 The manufacturing method according to claim 1, wherein the crystal to be manufactured is a coated crystal coated with the gel.
  9. 　生体物質の結晶を凍結する凍結工程を含む凍結結晶の製造方法であって、
    　請求項８記載の方法により前記被覆結晶を製造する被覆結晶製造工程をさらに含み、
    　前記凍結工程において、前記被覆結晶を凍結することを特徴とする製造方法。     A method for producing a frozen crystal comprising a freezing step of freezing a crystal of biological material,
    The method further comprises a coated crystal production step of producing the coated crystal by the method according to claim 8,
    In the freezing step, the coated crystal is frozen.
  10. 　前記凍結工程に先立ち、前記被覆結晶を抗凍結剤に浸漬させる浸漬工程をさらに含む請求項９記載の製造方法。 The manufacturing method according to claim 9, further comprising a dipping step of dipping the coated crystal in an antifreezing agent prior to the freezing step.
  11. 　種結晶である生体物質結晶を前記生体物質の溶液中でさらに成長させる結晶成長工程を含む、前記生体物質の結晶の製造方法であって、
    　前記結晶成長工程において、前記種結晶が、請求項８記載の方法により製造される被覆結晶であることを特徴とする製造方法。     A method for producing a crystal of a biological material, comprising a crystal growth step of further growing a biological material crystal that is a seed crystal in a solution of the biological material,
    The manufacturing method according to claim 8, wherein the seed crystal is a coated crystal manufactured by the method according to claim 8.
  12. 　請求項１から１１のいずれか一項に記載の製造方法により製造される結晶。 A crystal produced by the production method according to any one of claims 1 to 11.
  13. 　生体物質の結晶を構造解析する方法であって、
    　請求項８記載の製造方法により製造された被覆結晶または請求項９もしくは１０記載の製造方法により製造された凍結結晶を構造解析する構造解析工程を含むことを特徴とする構造解析方法。     A method for structural analysis of a crystal of a biological material,
    A structural analysis method comprising a structural analysis step of analyzing a structure of a coated crystal manufactured by the manufacturing method according to claim 8 or a frozen crystal manufactured by the manufacturing method according to claim 9 or 10.
  14. 　前記構造解析工程において、Ｘ線結晶構造解析または中性子線結晶構造解析により前記結晶を構造解析する請求項１３記載の構造解析方法。 14. The structural analysis method according to claim 13, wherein in the structural analysis step, the crystal is subjected to structural analysis by X-ray crystal structure analysis or neutron beam crystal structure analysis.
  15. 　生体物質の結晶化条件をスクリーニングする結晶化スクリーニング方法であって、
    　前記生体物質の結晶を製造する結晶製造工程と、
    　前記結晶製造工程における結晶化条件をスクリーニングするスクリーニング工程を含み、
    　前記結晶製造工程において、請求項１記載の結晶製造方法または請求項９記載の凍結結晶製造方法により前記結晶を製造することを特徴とする結晶化スクリーニング方法。     A crystallization screening method for screening crystallization conditions of a biological material,
    A crystal production process for producing crystals of the biological material;
    Including a screening step of screening crystallization conditions in the crystal manufacturing step,
    A crystallization screening method, wherein, in the crystal production step, the crystal is produced by the crystal production method according to claim 1 or the frozen crystal production method according to claim 9.
  16. 　生体物質の結晶化条件をスクリーニングする結晶化スクリーニング装置であって、
    　前記生体物質の結晶を製造する結晶製造手段と、
    　前記生体物質の結晶化条件をスクリーニングするスクリーニング手段とを含み、
    　前記結晶製造手段が、ゲル中で前記生体物質の結晶を析出させる結晶化手段を含むことを特徴とする結晶化スクリーニング装置。     A crystallization screening apparatus for screening crystallization conditions of a biological material,
    Crystal manufacturing means for manufacturing crystals of the biological material;
    Screening means for screening the crystallization conditions of the biological material,
    The crystallization screening apparatus, wherein the crystal production means includes a crystallization means for precipitating crystals of the biological material in a gel.
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