KR101845020B1 - Method for using nanoparticles as nucleation agents for the crystallization of proteins - Google Patents

Method for using nanoparticles as nucleation agents for the crystallization of proteins Download PDF

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KR101845020B1
KR101845020B1 KR1020160037077A KR20160037077A KR101845020B1 KR 101845020 B1 KR101845020 B1 KR 101845020B1 KR 1020160037077 A KR1020160037077 A KR 1020160037077A KR 20160037077 A KR20160037077 A KR 20160037077A KR 101845020 B1 KR101845020 B1 KR 101845020B1
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nanoparticles
protein
crystallization
gold nanoparticles
acid
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KR20170041611A (en
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최정우
최인희
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서울시립대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G7/00—Compounds of gold
    • 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
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
    • G01N23/2055—Analysing diffraction patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G01N2001/4027—Concentrating samples by thermal techniques; Phase changes evaporation leaving a concentrated sample

Abstract

The present invention is Bacillus subtilis YesR, chicken egg white lysozyme having a, in particular various sizes and shapes of the his-tag at the amino terminus using gold nanoparticles having carboxyl groups relates to a protein crystallization methods using nanoparticles as a nucleation-inducing agent , bovine serum albumin, Alicyclobacillus acidocaldarius acetyl-CoA carboxylase, and Listeria monocytogenes hypothetical protein protein and Ni 2+ ion were crystallized using gold nanoparticles as compared to the control without gold nanoparticles By confirming that the crystallization success rate is increased and various crystallization conditions can be found, the nucleation induction method of the present invention can be usefully used for identifying the protein structure by increasing the protein crystallization success rate.

Description

[0001] The present invention relates to a protein crystallization method using nanoparticles as nucleation inducers,

The present invention relates to a method for crystallizing a protein by using nanoparticles as a nucleation inducer. More specifically, the present invention relates to a method for crystallizing a protein by attaching a His-tag to a protein and using a Ni 2+ ion as a linker to form a carboxyl group to a nanoparticle having a carboxyl group to induce nucleation of the protein and increase the possibility of crystallization.

X-ray crystallography is a method of structural biology that provides important clues to understand the function of biopolymers, including proteins, through their structure, and 89% of the biopolymers known to date are synthesized by X-ray crystallography It was decided. Structural determination using X-ray crystallography consists of several steps such as mass expression of proteins, purification, crystallization, X-ray diffraction data collection, and computer-assisted structure identification. In order to crystallize a protein, it is necessary to make the protein supersaturated. It is possible to increase the concentration of the protein or increase the concentration of the reagent that induces the precipitation of the protein, thereby inducing supersaturation of the protein (FIG. 1). However, the concentration of the protein and the precipitating solution must be precisely enough to induce nucleation. If the concentration is too high, atypical precipitation will occur. If the concentration is too low, it will remain at the metastable state, and no precipitation will occur. The formation of spontaneous protein crystals is divided into the formation of nuclei and the growth of crystals (Fig. 2). In order to form a stable nucleus, proteins that existed in a single body in a solution must be gathered to form a lump larger than a certain size. Once nuclei are formed, the growth of the crystals lasts while the protein remains supersaturated. Several factors have been studied as factors affecting the crystallization of proteins, including precipitants, pH, temperature, pressure, and gravity, but the crystallization conditions of each protein are different (Adachi, H. et al., Temperature-screening system for determining protein crystallization conditions. Jpn. J. Appl. Phys., 44, 4080-4083, 2005), and thus the crystallization process remains a process that must undergo trial and error by attempting many conditions. The formation of nuclei is recognized as the most important step during the crystallization process, and thus inducing the formation of nuclei is an important method for increasing the success rate of protein crystallization.

Many advances have been made at various stages of X-ray crystallography through the advancement of modern biology and technology, but the crystallization phase of proteins remains a time-consuming or unsuccessful step due to repeated trial and error.

Accordingly, the present invention provides a method of using nanoparticles to help nucleation processes, which play an important role in protein crystallization. In previous studies, induction of nucleation using gold nanoparticles has been reported previously, but it was a non-selective method that utilizes the binding of gold particles and sulfur derived from cyanide or methionine exposed on the surface of proteins. Therefore, it can be applied only to proteins having a sulfur element exposed to the surface, and it is also difficult to form a high purity nucleus which is useful for crystallization by binding to a protein mixed with an impurity other than a target protein. Thus, the present invention has been completed by demonstrating that the protein crystallization method of the present invention can be effectively used as a nucleation method with high purity for increasing the protein crystallization success rate.

It is an object of the present invention to provide a protein crystallization method using nanoparticles as a nucleation inducer.

In order to achieve the above object, the present invention provides a method for crystallizing a protein, comprising the step of mixing carboxyl-coated nanoparticles and a protein having a his-tag in the presence of Ni 2+ ions, nucleation induction method.

The present invention also provides a method for inducing selective binding of protein orientation for crystallization of a protein comprising mixing carboxyl-coated nanoparticles and a protein having a his-tag in the presence of Ni 2+ ions.

Further, according to the present invention,

1) Nanoparticle mixed resin coated with a carboxyl group containing Ni 2+ ; And

2) a kit for crystallizing a protein containing a protein having a His-tag on its surface.

The present invention also provides a nucleation inducer for protein crystallization, comprising carboxyl-coated nanoparticles coated with nanoparticles having a carboxyl group.

Crystallization of proteins is an essential step in X-ray crystallography, but it remains difficult and time-consuming to predict. Induction of nucleation using gold nanoparticles has the effect of increasing the success rate of crystallization by helping to occur the most important process in the crystallization process. Increasing the success rate of crystallization shortens the time for structural identification and also makes it possible to study the structure of proteins that have failed crystallization in the past and can not reveal the structure.

The present invention, unlike the method using a combination of sulfur and resulting from the cysteine or methionine exposed on the surface of gold particles and the protein used in the conventional references are denoted by the His-tag of the protein using Ni 2+ ion to the linker The nanoparticles bind to the nanoparticles having a carboxyl group, and the nanoparticles bind strongly and rapidly, and the nanoparticles selectively bind only to a protein having a his-tag to induce nucleation, and a portion having a his- The orientation of the protein to the nanoparticles can be controlled. In addition, by studying the influence of the size, shape, and concentration of nanoparticles on nucleation for protein crystallization in the present invention, the protein crystallization method using nanoparticles as a nucleation inducer of the present invention can improve the protein crystallization success rate, As shown in FIG.

1 is a diagram showing a phase change of a protein according to the concentration of a crystallization inducing compound and a protein.
Fig. 2 is a diagram showing a step in which a protein monolith must go through to form crystals. Fig.
FIG. 3 is a view showing how a gold nanoparticle coated with a carboxyl group binds to a protein having a his-tag in the presence of Ni 2+ ions.
FIG. 4 is a graph showing that gold nanoparticles can induce nucleation for crystallization by collecting a protein having His-tag through the bond shown in FIG.
FIG. 5 is a graph showing that gold nanoparticles of different sizes can achieve various forms of nucleation favoring crystallization. FIG.
FIG. 6 is a graph showing the relative sizes and shapes of gold nanoparticles used for protein crystallization. FIG.
7 is a view showing various methods of coating a surface of the nanoparticles so as to have a carboxyl group group.
8 is a view showing the structure of a 96-well sitting drop plate used for protein crystallization.
9 and 10 are graphs showing the compositions of 96 kinds of MCSG-2T crystallization solutions used for protein crystallization.
Fig. 11 is a graph showing the conditions under which crystals were formed when gold nanoparticles of various sizes and shapes were used together with the control group without gold nanoparticles as a result of inducing crystallization of Bacillus subtilis YesR protein in 96 kinds of MCSG-2T crystallization solutions.
12 is a diagram showing crystals formed in the presence of gold nanoparticles as a result of protein crystallization.
FIG. 13 is a graph showing the conditions under which crystals were formed when gold nanoparticles having various sizes and shapes were used together with a control without gold nanoparticles as a result of crystallization of chicken egg white lysozyme protein in 96 kinds of MCSG-2T crystallization solutions.
Fig. 14 is a graph showing the conditions under which crystals were formed when gold nanoparticles of various sizes and shapes were used together with the control group without gold nanoparticles as a result of bovine serum albumin protein crystallization in 96 kinds of MCSG-2T crystallization solutions.
FIG. 15 is a graph showing the conditions under which crystals were formed when various sizes and shapes of gold nanoparticles were used together with the control group without gold nanoparticles as a result of crystallization of alicyclobacillus acidocaldarius acetyl-CoA carboxylase protein in 96 kinds of MCSG-2T crystallization solutions.
FIG. 16 is a graph showing the conditions under which crystals were formed when gold nanoparticles of various sizes and shapes were used together with a control group without gold nanoparticles as a result of crystallization of listeria monocytogenes hypothetical protein in 96 kinds of MCSG-2T crystallization solutions.
FIG. 17 is a graph showing the conditions under which crystals were formed when a chicken egg white lysozyme protein crystallized in 96 kinds of MCSG-2T crystallization solutions and a control group without gold nanoparticles and two star-shaped gold nanoparticles were used together.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for inducing nucleation for crystallization of proteins, comprising mixing carboxyl-coated nanoparticles and a protein having a his-tag in the presence of Ni 2+ ions.

The nanoparticles can be fabricated in various compositions, sizes, and shapes, and platinum nanoparticles, silver nanoparticles, magnetic nanoparticles, polymer nanoparticles, and gold nanoparticles can all be formed. In a preferred embodiment of the present invention, And is spherical, rod-shaped and star-shaped, and has a diameter of 5 to 100 nm in the case of a spherical shape, a width of 36 nm to 55.5 nm and a length of 15 nm in the case of a rod shape, a diameter of 15 mm To 230 mm.

The number of the nanoparticles is preferably 10 3 to 10 7 , more preferably 10 4 , per crystallization condition.

The nanoparticles are preferably coated with a carboxyl group on the surface of the nanoparticles using organic molecules rich in carboxyl groups. The organic molecules rich in carboxyl groups include citrate, polyacrylic acid, glutathione (Glutathione, GSH ), Thioglycolic acid (TGA), cysteine, mercaptobenzoic acid, D-penicillamine, dihydrolipoic acid (DHLA) and the like , And most preferred is a citrate according to a preferred embodiment of the present invention.

When using the citric acid salt in the process of synthesizing nanoparticles with a surface stabilizer, nanoparticles surface is covered with a carboxyl group, a carboxyl group is Ni 2+ ion and Ni 2+ ion is combined with a protein and coupled with the back-his tag (Fig. 3). Proteins with His-tag are frequently used for purification purposes, and binding of these carboxyl groups, Ni 2+ ions, and his tag sequence provides a selective and strong method of binding proteins and nanoparticles. This provides several advantages over non-selective methods that utilize the combination of conventional gold particles with sulfur derived from cysteine or methionine on the surface of the protein. First, The other proteins that are mixed with impurities are excluded. Secondly, the directions of binding proteins can be adjusted by changing the position of his-tag and the orientation of the proteins bound to the nanoparticles are relatively constant. Finally, Nanoparticles can be used by surface coating with carboxyl groups on nanoparticles having various compositions and shapes.

The nanoparticle synthesis process may include additional surface treatment steps, and it is possible to use surface treatment methods such as PAA, GSH, TGA, cysteine, mercaptobenzoic, D-penicillamine and DHLA.

The binding of a protein having a his-tag and a nanoparticle of the present invention has an effect of collecting proteins around the nanoparticles, and the proteins directly bound to the nanoparticles form a bond with other proteins, (Fig. 4). Analysis of the crystal structures of proteins that have already been identified reveals that they are deposited in a different way when crystals are formed and the binding of nanoparticles and proteins can be done in different ways depending on the size and shape of the nanoparticles, Are similar to proteins and provide different curvatures depending on the size and shape of the nanoparticles (Figure 5). Thus, the use of different types of nanoparticles can increase the chance of making nuclei with the shape most suitable for the crystallization of specific proteins.

The protein is preferably a protein with his-tag have about 10 ㎎ / ㎖ concentration, the nanoparticles and the protein is 1: 10 7 To 1: 10 < 13 & gt ;, more preferably 1: 10 < 10 >

In a specific embodiment of the present invention, the present inventors synthesized gold nanoparticles having various sizes and shapes having carboxyl groups, and using these, Bacillus subtilis YesR, chicken egg white lysozyme, bovine serum albumin, Alicyclobacillus As a result of crystallization under conditions of acidocaldariac acetyl-CoA carboxylase and Listeria monocytogenes hypothetical protein protein and Ni 2+ ion, crystallization with gold nanoparticles increased the success rate of crystallization compared with the control without gold nanoparticles , It is confirmed that various crystallization conditions can be found. Thus, the nucleation induction method of the present invention can be usefully used for protein structure identification by increasing the protein crystallization success rate.

The present invention also provides a method for inducing protein orientation for crystallization of proteins, comprising mixing carboxyl-coated nanoparticles and a protein having a his-tag in the presence of Ni 2+ ions.

The nanoparticles can be fabricated in various compositions, sizes, and shapes, and platinum nanoparticles, silver nanoparticles, magnetic nanoparticles, polymer nanoparticles, and gold nanoparticles can all be formed. In a preferred embodiment of the present invention, And is spherical, rod-shaped and star-shaped, and has a diameter of 5 to 100 nm when it is spherical, 36 to 55.5 nm and 15 nm when it is rod-shaped, and 15 nm to 230 nm.

The number of the nanoparticles is preferably 10 3 to 10 7 , more preferably 10 4 , per crystallization condition.

The nanoparticles are preferably coated with a carboxyl group on the surface of the nanoparticles by using organic molecules rich in carboxyl groups. The carboxyl-rich organic molecules include citrate, polyacrylic acid, glutathione (GSH) ), Thioglycolic acid (TGA), cysteine, mercaptobenzoic acid, D-penicillamine, dihydrolipoic acid (DHLA) and the like , And most preferred is a citrate according to a preferred embodiment of the present invention.

The nanoparticle synthesis process may include additional surface treatment steps, and it is possible to use surface treatment methods such as PAA, GSH, TGA, cysteine, mercaptobenzoic, D-penicillamine and DHLA.

The protein is 10 ㎎ / ㎖ preferably a protein with his-tag with the concentration, the nanoparticles and the protein is 1: 10 7 to 1: 10, 13 number ratio is desirable, and one of: the number of 10 10 Is more preferable.

Bacillus subtilis YesR, chicken egg white lysozyme, bovine serum albumin, Alicyclobacillus acidocaldarius acetyl-CoA carboxylase, and Listeria monocytogenes hypothetical protein having a his-tag at the amino terminus using gold nanoparticles of various sizes and shapes having carboxyl groups As a result of crystallization under protein and Ni 2+ ion conditions, the crystallization rate using gold nanoparticles increased as compared with the control without gold nanoparticles, confirming that various crystallization conditions can be found , The protein orientation induction method of the present invention can be usefully used for protein structure identification by increasing protein crystallization success rate.

Further, according to the present invention,

1) Nanoparticle mixed resin coated with a carboxyl group containing Ni 2+ ; And

2) a kit for crystallizing a protein containing a protein having a His-tag on its surface.

The present invention also provides a nucleation inducer for protein crystallization, comprising carboxyl-coated nanoparticles coated with nanoparticles having a carboxyl group.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

≪ Example 1 > Synthesis of gold nanoparticles

The effect of gold nanoparticles on the crystallization of Bacillus subtilis YesR, chicken egg white lysozyme, bovine serum albumin, Alicyclobacillus acidocaldarius acetyl-CoA carboxylase, and Listeria monocytogenes hypothetical protein proteins with his-tag at the amino terminus 14 gold nanoparticles were synthesized as follows; Spherical nanoparticles with diameters of 5, 10, 15, 18, 20, 50 and 100 nm, 15 x 36 and 15 x 55.5 nm, diameters of 15, 30, 60, 125 and 230 nm Star nanoparticles were synthesized.

<1-1> Synthesis of spherical gold nanoparticles

In the case of spherical gold nanoparticles, a hydrothermal synthesis method was used in which HAuCl 4 precursor was used and sodium citrate was used as a surface stabilizer to reduce heat while being heated. The size of the nanoparticles can be controlled by adjusting the amount of the surface stabilizer, and the method of synthesizing the spherical nanoparticles of 20 nm is as follows.

A solution of 1 g of HAuCl 4 dissolved in 20 ml of distilled water and 1 g of sodium citrate in 100 ml of distilled water was prepared and then 0.2 ml of HAuCl 4 stock solution was added to 198.27 ml of distilled water and stirred until boiling And heated. When the solution began to boil, 1.53 ml of sodium citrate solution was added, and the color changed from pale yellow to dark gray. After sufficiently reducing, the color changed to purple. At this time, the heating was stopped and the solution was cooled at room temperature.

<1-2> Synthesis of rod-shaped gold nanoparticles

In the case of the production of rod-shaped gold nanoparticles, small gold nanoparticles were prepared by using HAuCl 4 as a precursor, cetyltrimethylammonium bromide (CTAB) as a surface stabilizer and sodium borohydride (NaBH 4 ) as a reducing agent Seed-mediated growth method in which silver ions and ascorbic acid are used for growth. The detailed synthesis method is as follows.

5 nm spherical gold nanoparticles were prepared in one step. 100 mM HAuCl 4 storage solution and 10 mM NaBH 4 solution were prepared, and the NaBH 4 solution was activated at 0 ° C for 1 hour or more. To 40 ml of distilled water, 2.2 g of CTAB was added, 100 μl of HAuCl 4 storage solution was added to the solution, and the mixture was stirred for an additional 1 hour. After that, 2.4 mL of NaBH 4 solution was added, and the mixture was agitated for 2 minutes and then allowed to stand at room temperature for 3 hours or more.

The growth solution was prepared by adding 3.64 g of CTAB to 99.5 ml of distilled water, adding 500 μl of the HAuCl 4 storage solution, and further stirring. Then, add 1 ml of a solution of 0.03 g AgNO 3 in 25 ml of distilled water, add 700 μl of ascorbic acid (0.24 g), which acts as a weak reducing agent, in 10 ml of distilled water, and add trivalent gold ions 0 is reduced and changed to transparent.

80 μl of the gold nanoparticle solution prepared in the first step is added to the growth solution. When the solution is left overnight without any impact or stimulation, 5 nm gold nanoparticles grow to become the gold nanoparticles, and CTAB The length of the gold nanoparticles can be controlled by controlling the amount of AgNO 3 .

<1-3> Synthesis of star-shaped gold nanoparticles

In the case of star-shaped gold nanoparticles, seed-mediated growth method was adopted in which spherical gold nanoparticles were grown as seeds. At this time, HAuCl 4 was used as a precursor, sodium citrate was used as a surface stabilizer, and monodisperse nanoparticle solutions were further synthesized using glycerol. The detailed synthesis method is as follows.

First, spherical gold nanoparticle solutions with diameters of 5, 10, 20, 40, 50, and 100 nm were prepared. As described above, a hydrothermal synthesis method using sodium citrate as a surface stabilizer was used as a precursor of HAuCl 4 , and the size of the gold nanoparticles was adjusted by varying the amount of the surface stabilizer. The resulting gold nanoparticle solution was cooled at room temperature and used as a seed solution for the synthesis of star-shaped gold nanoparticles.

Next, add 300 μl of gold nanoparticle seed solution and 1 ml of glycerol to 9.8 ml of distilled water, and stir well. After 5 minutes, quickly add 22 μl of 1% sodium citrate, 100 μl of 1% HAuCl 4 and 42.5 μl of 0.1% AgNO 3 in a growth solution. Then immediately add 100 μl of 1% hydroquinone solution. Thereafter, when the stirrer is immediately stopped, the color of the solution changes from red to blue within a few minutes.

Therefore, spherical gold nanoparticles and star-shaped gold nanoparticles were synthesized using a surface stabilizer having a carboxyl group, so that they could be used without further surface treatment. In the case of the rod-shaped gold nanoparticles, CTAB (Cetyltrimethylammonium bromide ), Which was synthesized by using a surface stabilizer. Thus, PAA (polyacrylic acid) rich in carboxyl group was used for coating (FIG. 7). In addition to the coating using PAA, nanoparticles can be prepared using various surface treatment methods shown in FIG.

 As a result, as shown in FIG. 6, spherical gold nanoparticles having diameters of 5, 10, 15, 18, 20, 50 and 100 nm and rod-shaped gold nanoparticles having a size of 15 x 36 and 15 x 55.5 nm , And star-shaped gold nanoparticles having diameters of 15, 30, 60, 125, and 230 nm were synthesized (FIG. 6).

< Experimental Example  1> Using gold nanoparticles Bacillus subtilis YesR  Confirm protein crystallization

The gold nanoparticles synthesized in Examples <1-1> and <1-2> were used to crystallize the Bacillus subtilis YesR protein having his-tag at the amino terminus.

Specifically, Bacillus subtilis YesR protein at a concentration of 5.6 mg / ml was dissolved in a buffer consisting of 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 1 mM NiCl 2 , and the protein crystallized using a 96-well sitting drop plate (Fig. 8). As shown in FIG. 9 and FIG. 10, 70 μl of MCSG 2T (96 conditions), a crystallization solution sold by microlytic, was added to each well and 0.5 μl of crystallization solution and 0.5 μl of protein solution containing 10 4 gold nanoparticles Were added to the drop and sealed with a transparent tape (Figs. 9 and 10). The plate was stored at 20 ℃ and the formation of crystals was observed by a microscope.

As a result, as shown in FIG. 11, the gold nanoparticles having a diameter of 5, 10, 15, 18, 20, 50 and 100 nm and the gold nanoparticles having a diameter of 15 x 36 and rod-shaped gold nanoparticles with a size of 15 x 55.5 nm) were added to the 96-well plate. In FIG. 10, the blue portion represents the case where crystals are formed in both the control group and the gold nanoparticle-containing region. The red region represents a crystal when gold nanoparticles are present, and the yellow region represents a crystal region only in the control group 11). The nine gold nanoparticles exhibited different crystallization conditions, and all of the crystallization conditions obtained using the nine gold nanoparticles were found to have 25 new crystallization conditions (total of 38 crystallization conditions). This indicates that the gold nanoparticles significantly increased the crystallization success rate of the Bacillus subtilis YesR protein compared to the 13 crystallization conditions in the control group, which is considered to be due to nucleation required for crystallization. In addition, it was confirmed that the protein crystals grow in a needle shape, a square shape, etc. as shown in FIG. 12 (FIG. 12).

<Experimental Example 2> Confirmation of protein crystallization using gold nanoparticles

Using the gold nanoparticles synthesized in Examples <1-1> to <1-2>, four proteins having a his-tag at the amino terminus (chicken egg white lysozyme, bovine serum albumin, Alicyclobacillus acidocaldarius acetyl-CoA carboxylase, Listeria monocytogenes hypothetical protein).

The protein used was chicken egg white lysozyme (7.8 ㎎ / ㎖), Bovine serum albumin (13.4 ㎎ / ㎖), Alicyclobacillus acidocaldarius acetyl-CoA carboxylase (13.6 ㎎ / ㎖) and Listeria monocytogenes hypothetical protein (12.1 ㎎ / And these proteins were dissolved in a buffer composed of 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 1 mM NiCl 2 , and a 96-well sitting drop plate was used for protein crystallization (FIG. 8). As shown in FIGS. 9 and 10, 70 μl of MCSG 2T (96 conditions), a crystallization solution sold by microlytic, was added to each well and 0.5 μl of a crystallization solution and 0.5 μl of 2 × 10 7 / ml of gold nanoparticles Of the protein solution was added to make 1.0 [micro] l drop, so that 10,000 gold nanoparticles per drop were added, followed by sealing with a transparent tape (Figs. 9 and 10). The plate was stored at 20 ℃ and the formation of crystals was observed by a microscope.

As a result, as shown in FIG. 13-16, the control group without the gold nanoparticles and the gold nanoparticles (spherical gold nanoparticles having diameters of 5, 10, 20, 50 and 100 nm and 15 x 36 And rod-shaped gold nanoparticles with a size of 15 x 55.5 nm) were added to the 96-well plate. In FIG. 13-16, the blue portion represents the case where crystals are generated in both the control group and the gold nanoparticle-containing crystals. In the case where the crystal is formed only when the gold nanoparticles are present in red, and when the crystal is formed only in the control group (Fig. 13-16). Seven gold nanoparticles exhibited different crystallization conditions and all of the new crystallization conditions obtained using the seven gold nanoparticles were combined with chicken egg white lysozyme, bovine serum albumin, Alicyclobacillus acidocaldarius acetyl-CoA carboxylase, and Listeria monocytogenes hypothetical protein In the case of the four proteins, in the control group, 18, 6, 3, and 2 crystallization conditions were found, respectively. When gold nanoparticles were added, 19, 2, 8 and 2 new crystallization conditions were found , 8, 11, and 4 kinds of crystallization conditions). This indicates that the gold nanoparticles significantly increased the crystallization success rate of the protein.

As shown in the above experiment example, the addition of gold nanoparticles showed the effect of increasing the crystallization conditions of the five proteins used in the experiment examples, which can increase the possibility of crystallization by inducing nucleation of protein Lt; / RTI &gt;

Experimental Example 3 Confirmation of protein crystallization using star-shaped gold nanoparticles

Crystallization of the chicken egg white lysozyme having his-tag at the amino terminus was further performed using the star-shaped gold nanoparticles synthesized in Example 1-3.

Specifically, the protein used was dissolved in a buffer consisting of 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 1 mM NiCl 2 in chicken egg white lysozyme (7.8 mg / ml) drop plate was used (Fig. 8). As shown in FIGS. 9 and 10, 70 μl of MCSG 2T (96 conditions), a crystallization solution sold by microlytic, was added to each well and 0.5 μl of a crystallization solution and 0.5 μl of 2 × 10 7 / ml of gold nanoparticles Of the protein solution was added to make 1.0 [micro] l drop, so that 10,000 gold nanoparticles per drop were added, followed by sealing with a transparent tape (Figs. 9 and 10). The plate was stored at 20 ℃ and the formation of crystals was observed by a microscope.

As a result, as shown in FIG. 17, the presence of crystals was confirmed in a 96-well plate to which gold nanoparticle-free control and two star-shaped gold nanoparticles (diameters of 30 and 125 nm) were added. In FIG. 17, the blue portion represents the case where crystals are formed in both the control group and the gold nanoparticles, and the red represents the case where crystals are formed only when gold nanoparticles exist, while the yellow represents crystals only in the control group 17). The two gold nanoparticles showed different crystallization conditions, and when all of the crystallization conditions obtained using the two gold nanoparticles were combined, 25 new crystallization conditions could be found. This indicates that the gold nanoparticles significantly increased the crystallization success rate of the protein compared to the 18 crystallization conditions in the control group without the gold nanoparticles.

Claims (13)

Citric acid, citric acid, poly acrylic acid, glutathione (GSH), thioglycolic acid (TGA), cysteine, mercaptobenzoic acid, D-penicillamine (D-penicillamine) and dihydrolipoic acid (DHLA), a nanoparticle having nanoparticles whose surface is coated with a carboxyl group and a protein having a his-tag using a carboxyl-rich organic molecule In the presence of Ni &lt; 2 + &gt; ions in the presence of Ni &lt; 2 + &gt; ions.
The method according to claim 1, wherein the nanoparticles are any one selected from the group consisting of platinum nanoparticles, silver nanoparticles, magnetic nanoparticles, polymer nanoparticles, and gold nanoparticles. Way.
The method of claim 2, wherein the nanoparticles are gold nanoparticles.
delete delete The method according to claim 1, wherein the nanoparticles are selected from the group consisting of spherical, rod-shaped, and star-shaped nanoparticles.
The nanoparticle according to claim 6, wherein the nanoparticles have a diameter of 5 to 100 nm when the nanoparticles are spherical, a width of 36 to 55.5 nm and a length of 15 nm when the nanoparticles have a rod shape, and a diameter of 15 to 230 nm when the nanoparticles are star- To induce nucleation of the protein.
The method according to claim 1, wherein 10 3 to 10 7 nanoparticles are used for protein crystallization.
The method according to claim 1, wherein the protein is any one selected from the group consisting of all proteins having his-tag.
The method of claim 1, wherein the nanoparticle and the protein are mixed in a ratio of 1:10 To about 1: 10 &lt; 13 &gt;. &Lt; / RTI &gt;
Citric acid, citric acid, poly acrylic acid, glutathione (GSH), thioglycolic acid (TGA), cysteine, mercaptobenzoic acid, D-penicillamine (D-penicillamine) and dihydrolipoic acid (DHLA), a nanoparticle having nanoparticles whose surface is coated with a carboxyl group and a protein having a his-tag using a carboxyl-rich organic molecule In the presence of Ni &lt; 2 + &gt; ions.
1) It is possible to use citrate, poly acrylic acid, glutathione (GSH), thioglycolic acid (TGA), cysteine, mercaptobenzoic acid, Wherein the surface of the nanoparticles is coated with a carboxyl group using a carboxyl-rich organic molecule selected from the group consisting of D-penicillamine, dihydrolipoic acid (DHLA), Ni 2+ -containing Nanoparticle mixing resin; And
2) A kit for protein crystallization containing a protein having a His-tag on its surface.
delete
KR1020160037077A 2015-10-07 2016-03-28 Method for using nanoparticles as nucleation agents for the crystallization of proteins KR101845020B1 (en)

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Title
Diana de Oliveira Ribeiro,‘Using Gold Nanoparticles in Protein Crystallography:Studies in crystal growth and derivatization’, FCT-UNL 석사학위 논문(2012)*
NANOPROBES E-NEWS, Vol 10, No. 7(2009.07.25.)*

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