WO2007023915A1 - Imprint polymer and use thereof - Google Patents

Imprint polymer and use thereof Download PDF

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Publication number
WO2007023915A1
WO2007023915A1 PCT/JP2006/316642 JP2006316642W WO2007023915A1 WO 2007023915 A1 WO2007023915 A1 WO 2007023915A1 JP 2006316642 W JP2006316642 W JP 2006316642W WO 2007023915 A1 WO2007023915 A1 WO 2007023915A1
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protein
metal
complex
polymer
target protein
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PCT/JP2006/316642
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French (fr)
Japanese (ja)
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Toshifumi Takeuchi
Takayuki Hishiya
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Japan Science And Technology Agency
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Priority to JP2007532181A priority Critical patent/JP5249582B2/en
Publication of WO2007023915A1 publication Critical patent/WO2007023915A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/52—Sorbents specially adapted for preparative chromatography
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/36—Amides or imides
    • C08F222/38—Amides
    • C08F222/385—Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

  • the present invention relates to a molecular imprint technique for specifically recognizing a target molecule.
  • the present invention relates to a method for synthesizing an imprinted polymer capable of recognizing a protein, and a method for using the method.
  • Amino acids constituting proteins include several residues that are easily metal-coordinated. Residues such as histidine, tributophan, and cysteine are amino acid residues having an unshared electron pair, and form efficient coordination bonds to transition metals such as copper and nickel. Therefore, these transition metals and ligands that capture metals firmly by the chelate effect A polypeptide may be recognized by the combination of
  • Molecular imprinting is a method for obtaining a polymer having a recognition site complementary to a target molecule. Details are as follows. First, the target molecule is a cage molecule, and a complex of the cage molecule and a functional monomer that can bind to the cage molecule is formed. Then, the obtained composite is polymerized with a crosslinking agent to obtain a crosslinked polymer. After that, by removing the cage molecule with a solvent or the like, a recognition site complementary to the target molecule is constructed in the polymer.
  • Minoura et al. Also imprinted glucose oxidase using silica gel as a support (Non-patent Document 2: M. Burow and N. Minoura, Biochem. Biophys. Res. Commun. 1996). , 227, 419-422). Positively charged 2- (dimethylamino) ethyl metatalylate, negatively charged acrylic acid, and glucosidase base glucosyl glucosylase in the side chain, charged In addition, acrylamide and four types of functional monomers are used to synthesize imprinted polymer that recognizes vertical molecules mainly through three types of interactions.
  • the imprint polymer produced by the conventional method has problems in terms of bond selectivity and applicability as a sensor, sensor array, or sensor chip.
  • an implementation technology that provides high substrate selectivity is required.
  • the present invention has been made in view of the above-described conventional problems, and an object of the present invention is to provide an imprint polymer with higher binding specificity and to provide an imprint polymer sensor by dye modification. Another object of the present invention is to provide a microarray that is a typical method of use. In addition, in order to overcome the low selectivity that has been a drawback of conventional imprinting, it is an object to improve specificity and suppress non-specific interactions due to the multivalent coordination unique to metal complexes.
  • An imprint polymer having at least one of a metal complex and a substrate inhibitor at a recognition site has been found to have higher selectivity than a conventional imprint polymer, and the present invention has been completed.
  • the present invention has been made in view of the above-described conventional problems, and includes the following inventions.
  • An imprint polymer comprising a recognition site that reversibly binds to a target protein, wherein the recognition site has at least one of a metal complex and a substrate inhibitor.
  • [0020] comprising a complex forming step of forming a complex of the target protein and the functional monomer, and a polymerization step of polymerizing the functional monomer in the complex, wherein the functional monomer is a target
  • a method for producing an imprinted polymer comprising a ligand coordinated to a metal capable of binding to a protein and a substrate inhibitor of a target protein
  • the method for producing an imprint polymer comprising the step of modifying the function of the imprint polymer by detaching the ligand force metal and then coordinating a metal different from the metal. .
  • a step of complexing a substrate-protein complex obtained by complexing a target protein and a substrate of the target protein and a functional monomer, and a step of polymerizing the functional monomer after the above step A protein immobilization method characterized by comprising:
  • FIG. 1 is a drawing showing a method for producing an imprint polymer in the present invention.
  • FIG. 2 is a structural formula of a complex according to an embodiment of the present invention.
  • FIG. 3 is a drawing showing a structural formula of a dye-modified monomer according to an embodiment of the present invention, where (a) shows a styrene-modified monomer and (b) shows a cyclen-modified monomer.
  • FIG. 4 is a drawing showing the structural formula of a covalent bond (tol) of a dye-modified complex-styrene monomer according to an embodiment of the present invention, where (a) is ethylenediamine-anthracene, and (b) is cyclen. Anthracene, (c) is a cyclen-dansyl, and (d) is an ethylenediamine-dansinore modified.
  • FIG. 5 is a drawing showing a structural formula of a styrene-modified ribonuclease inhibitor according to an embodiment of the present invention.
  • FIG. 6 is a drawing showing a synthesis scheme of a modified cyclone according to an example of the present invention.
  • FIG. 7 is a drawing showing a synthesis scheme of modified ethylenediamine according to an example of the present invention.
  • FIG. 8 is a drawing showing a synthesis scheme of styrene-modified phosphate UMP according to an example of the present invention.
  • FIG. 9 is a sensorgram for ribonuclease A of imprinted polymer and non-imprinted polymer according to an example of the present invention.
  • FIG. 10 is a chromatogram of protein separation using hydrophobic chromatography in an example of the present invention.
  • FIG. 12 Myoglobin (Al), ribonuclease A (A2), lysozyme (A3), albumin (A4), cytochrome C (A5), lactalbumin for five polymers prepared using Cu (cyc)
  • FIG. 13 is a drawing showing time-dependent changes in the refolding rate when LY-Cu (en) IP and Cu (en) -MIP are not added and when they are added.
  • FIG. 14 is a graph showing the change over time in the elution amount of lysozyme in the flow type reactivation method.
  • FIG. 15 is a sensorgram in which the binding affinity is lowered by adding EDTA and then the activity is restored by adding copper ion.
  • FIG. 16 Draft showing relative affinities of imprinted polymers synthesized with functional ethylene diamine as a functional monomer for each protein.
  • FIG. 17 A graph showing the relative affinity of each imprinted polymer synthesized from bulky cyclen as a functional monomer with a blank polymer.
  • FIG. 18 The affinity of ribonuclease A for the three polymers, imprint polymer, blank polymer, and GEMA alone, is dependent on the salt concentration.
  • FIG. 19 shows an example of analyzing the binding of Zn (TCPP (Tetrakis (4-carboxyphenyl) porphine)) to a substrate on which ribonuclease A is immobilized using an SPR sensorgram.
  • Zn TCPP (Tetrakis (4-carboxyphenyl) porphine
  • FIG. 20 is a UV absorption spectrum when titrating Zn [TCPP] (Tetrakis (4-carboxyphenyl) porphine) against ribonuclease A.
  • TCPP Tetrakis (4-carboxyphenyl) porphine
  • FIG. 21 is a graph showing the relative affinity of each of the imprinted polymers synthesized using bulky Zn [TCPP] as a functional monomer and the blank polymer for each protein.
  • the imprint polymer according to the present invention has a recognition site that reversibly binds to a target protein, and the recognition site has at least one of a metal complex and a substrate inhibitor.
  • a metal complex and a substrate inhibitor can be appropriately changed depending on the target protein, and are not particularly limited.
  • composition of the imprint polymer is not particularly limited. And those containing, as a main component, a polymer obtained by polymerizing a functional monomer.
  • Examples of functional monomers include acrylamide, acrylic acid, darcosyloxychetyl methacrylate, 1-butyrimidazole, 4-butyrpyridine, 2- (dimethylamino) ethyl methacrylate, and 2-hydroxyethyl methacrylate.
  • Water-soluble monomers such as rate are preferred. Water-soluble monomers are preferred because proteins, which are in vivo substances, are preferably imprinted in buffers that are highly sensitive to pH and organic solvents.
  • Hydrogen-bonding monomers such as a hydroxyl group, an amino group, and a carboxyl group can form a hydrogen bond with a target protein. This hydrogen bond can supplement the interaction by metal coordination.
  • Carboxylic acid, quaternary ammonia, sulfonic acid, phosphoric acid, and the like can reinforce interaction by ionic bonds.
  • the functional monomer contains at least one of the following metal complex (1-3) and a substrate inhibitor (1-4). Details will be described later.
  • the imprint polymer of the present invention may contain a dye-modified monomer for protein detection.
  • Specific functional dyes include azobenzenes such as methyl orange and methyl red, quinones such as naphthoquinone and anthraquinone, dialelemethanes such as phenoxazine and malachite green, triarylmethanes, fluorescein, rhodamine B, etc.
  • styryl such as stilbene and stilbazole
  • spiropyran such as spirobenzopyran and spiroxazine
  • diarylethenic such as difurylethene and diceninoreethene
  • squarylium such as squarylium and croconium
  • quercetin and anthocyanin And other flavonoid functional pigments styryl such as stilbene and stilbazole
  • spiropyran such as spirobenzopyran and spiroxazine
  • diarylethenic such as difurylethene and diceninoreethene
  • squarylium such as squarylium and croconium
  • anthocyanin And other flavonoid functional pigments.
  • a fluorescent dye such as coumarin dansyl 'FITC' anthracene.
  • Possible detection methods include fluorescence spectrum change, fluorescence lifetime measurement, and fluorescence energy transfer.
  • the dye is introduced in the vicinity of a recognition site by simply copolymerizing a monomer having a polymerizable functional group introduced thereto, by adsorbing it to a polymerized high molecule, or by covalently bonding to the metal complex of (13) below. There are three ways.
  • FIG. 3 shows an example of a dye-modifying monomer that can be used in the present invention.
  • FIG. 4 shows the structural formula of a covalently bonded complex of a dye-modified complex and a styrene monomer that can be used in the present invention. These dye-modified monomers can detect protein binding based on changes in fluorescence wavelength peak and fluorescence lifetime.
  • the structures of the dye and the metal complex are not limited to those shown in the figure, and those shown above and those shown in the following (13) can be used.
  • the amino acids that make up a protein contain a number of ⁇ ⁇ residues that facilitate metal coordination.
  • Residues such as histidine, tributophan, and cysteine are amino acid residues having an unshared electron pair, and form efficient coordination bonds to transition metals such as copper and nickel.
  • the imprint polymer of the present invention has a metal complex containing these metal atoms at the recognition site.
  • a metal complex consists of a metal and a ligand that coordinates to the metal. Since the recognition site of the imprinted polymer according to the present invention has this metal complex, it can recognize these amino acid residues via the metal atom and recognize the target protein.
  • a metal complex is composed of a ligand and a metal that is coordinated to the ligand, but in this embodiment, the term “metal complex” is a state in which the ligand is alone ( That is, it may refer to both a state in which the metal is not coordinated and a state in which the metal is coordinated.
  • the imprint polymer according to the present invention recognizes a target molecule through a metal atom in this way, it can recognize a target protein under a wider range of conditions.
  • Li ribonuclease and its typical ribonuclease inhibitor (2, -CMP) and can not obtain a low P H unless high affinity around 5.5.
  • the imprint polymer of the present invention can bind to a target protein in a relatively wide range of pH, does not function in other protein-ligand interactions, and acquires affinity even under conditions. Can do.
  • the artificial receptor obtained by the present invention that is based only on pH, Binding is possible in a wide range of temperature and salt concentration.
  • the imprinted polymer of the present invention is capable of binding even under such conditions.
  • the construction of a recognition site using a metal complex has an advantage not found in nature.
  • stability is a concern during materialization!
  • the metal coordination can be selected from various combinations of the metal and the ligand.
  • metals can be desorb or recombine metals with the same ligand, and the function can be freely changed.
  • the metal can be dissociated by EDTA, and another metal can be introduced. It is also possible to increase the affinity by using a metal having a coordinating form of the same genus or to change the substrate selectivity by adding a metal having a different coordinating form.
  • Some lanthanide metals have peptide bond cleavage activity, which can be used to cleave target proteins.
  • transition metals such as copper, zinc, manganese, nickel, and iron
  • rare earth metals such as copper, zinc, manganese, nickel, and iron
  • rare earth metals such as rare earth metals, lanthanide metals that emit fluorescence, and the like
  • lanthanide metals that emit fluorescence, and the like can be used as the metal that coordinates with the ligand.
  • a polyvalent coordination complex such as ethylenediamine, cyclene, dipicolylamine, iminodiacetic acid, utilotriacetic acid, EDTA, and volphiline can be used.
  • FIG. 2 shows an example of a metal complex applicable to the present invention.
  • the functional monomer may be the ligand described in this column and the substrate inhibitor itself in the following (14) column, or recognizes the target protein in the ligand and the substrate inhibitor. It may be a compound with a polymerization functional group introduced at a position different from the site! /. Ligand and substrate inhibitor can be polymerized via this polymerization functional group to form an imprint polymer. In the present invention, whether or not a polymerization functional group is introduced is referred to as a ligand or a substrate inhibitor.
  • styrene or acrylic acid may be introduced into the ligand and the substrate inhibitor by, for example, an organic synthesis method.
  • the complex can be bonded to each other to form a binuclear complex as a functional monomer.
  • nonspecific binding can be suppressed by inactivating a metal complex site immobilized so that it cannot specifically bind to a protein using a chelating agent. it can.
  • a metal complex site immobilized immobilized so that it cannot specifically bind to a protein using a chelating agent. it can.
  • the metal complex that is immobilized by specifically interacting with the protein is involved in the recombination, and high substrate selectivity is expressed.
  • the substrate inhibitor is a compound having a structure capable of specifically binding at least a part to the substrate binding site of the target protein, like the substrate of the enzyme which is the target protein.
  • a substrate inhibitor must not be catalyzed by an enzyme, or it must have a very slow reaction rate even when catalyzed.
  • Such substances include competitive inhibitors having a structure similar to that of the target protein substrate, and pseudo-substrates. These substrate inhibitors have high specificity for binding to the target protein. Therefore, high target recognition characteristics can be imparted to the imprint polymer.
  • the binding of the target protein to the substrate recognition site is stable.
  • the affinity of the substrate inhibitor is not particularly limited, it is preferably 103M or more.
  • FIG. 5 shows the structural formulas of styrene-modified ribonuclease inhibitors (2, -UMP and 3, -UMP).
  • the imprint polymer of the present invention may be provided with such a substrate inhibitor alone or with the metal complex in the above-mentioned (13) column! By providing it in combination with a metal complex, the imprint polymer can obtain high affinity due to a cooperative effect.
  • the imprint polymer according to the present invention contains, as a functional monomer, at least one of the metal complex and the substrate inhibitor described in the above (11) and (1-2). Including comonomers that copolymerize with functional monomers.
  • the comonomer to be copolymerized with the functional monomer the functional monomer described in the above (11) can be used.
  • Addition of MPC ([2- (methacryloyloxy) ethyl] phosphorylcholine) or GEMA (Glycosyloxyethyl methacrylate) reduces non-specific interactions.
  • the method for producing an imprint polymer according to the present invention includes a complex forming step for forming a complex of a target protein and a functional monomer, and a polymerization step for polymerizing the functional monomer in the complex.
  • a target protein As shown in (a) and (b) of FIG. 1, a target protein, a metal capable of coordinating and binding to the target protein, and a metal complex that also has a ligand force to coordinate the metal. Then, they are associated via the metal (complex formation step). At this time, the polymer synthesis system includes V, not shown, and a polymerizable functional monomer.
  • the functional complex is polymerized to fix the metal complex at a position complementary to the target protein (polymerization step).
  • the target protein is removed.
  • a conventional method for producing an imprinted polymer can be suitably used as other conditions in which the target protein and the functional polymer are mixed.
  • the complex formation between the functional monomer and the protein substrate is preferably performed in water.
  • Particularly preferred conditions in the complex formation step are a protein concentration of several hundred M and a functional monomer of about 2 equivalents to 5 equivalents (in the case of ribonuclease).
  • the protein must be in a concentration range where it does not associate.
  • the functional monomer must be thicker than the dissociation constant and the concentration condition.
  • the temperature is room temperature.
  • the ligand of (1 3) above and the substrate of (1 4) above A functional monomer containing at least one of the inhibitors may be used.
  • the complex formation step and the following polymerization step may be performed in the presence of the comonomer (15) above.
  • the complex formation step and the polymerization step may be performed in the presence of a crosslinking agent!
  • a crosslinking agent a relatively water-soluble compound such as methylenebisacrylamide can be used. Further, those having no metal coordination ability are preferred.
  • the affinity should be about 10 3 M.
  • 2'-CMP is a relatively good inhibitor for ribonucleases.
  • a polymerizable functional group may be introduced at the 5 ′ position without being involved in the bond.
  • the analog of the phosphoric acid compound is very useful as a functional monomer.
  • the selectivity is usually very low, but by using this substrate inhibitor, strong interaction and selectivity can be given.
  • Affinity can be improved by combining with ionic monomers such as carboxylic acids, hydrogen bonding monomers such as pyridine, or interactions used in normal imprinting such as metal complexes.
  • the affinity and specificity for the target protein can be improved by immobilizing the substrate inhibitor in the polymerization step.
  • Peptide inhibitors for peptidases, sugar analogs for lectins, and phosphorylated peptides for various kinases correspond.
  • the amount of the functional monomer is preferably about 2 to 5 equivalents with respect to the target protein. If a substrate inhibitor is used as a monomer, it should be at least 1 equivalent to the target protein!
  • the buffer solution used may not be a buffer solution that inhibits metal coordination.
  • Phosphate buffer solution may be undesirable because it exhibits inhibitory activity against zinc complexes and the like.
  • the Tris buffer solution is more powerful than the HEPES buffer. Many cases where the coordination ability is low are preferred.
  • a carbonic acid boric acid / acetic acid / phosphate buffer solution can be used.
  • the polymerization reaction is a step of polymerizing the functional monomer in the complex formed in the complex formation step of (2-1) above by adding a polymerization initiator (sometimes referred to simply as an initiator). It is.
  • the polymerization method both polymerization by UV light and thermal polymerization are possible.
  • an azo compound-based initiator or a Redox initiator such as potassium persulfate or ammonium persulfate can be used.
  • TEMED which is a polymerization accelerator, the polymerization reaction proceeds even under mild conditions.
  • the complex formed in the complex forming step (2-1) is also isolated from the aqueous solution in which the complex forming step is performed, and then the polymerization step is performed.
  • the polymerization step in the case of using a metal complex can be carried out after the protein-metal complex complex is isolated, whereby a highly selective recognition material can be prepared.
  • Pd dissociation rate
  • binucleation of metal complexes is effective.
  • general separation methods such as gel filtration, HPLC, and dialysis can be used.
  • the complex can then be isolated by lyophilization.
  • selectivity can be improved by suppressing non-specific binding of the target protein.
  • the metal in the metal complex in the functional monomer (after the polymerization step and in the imprint polymer) can bind to this metal. Bind the ligand. If there is a metal complex that is precisely located at a position complementary to the saddle protein, non-specific binding occurs. According to the blocking agent, such a metal complex that is not accurately arranged can be blocked, so that specificity is improved.
  • a blocking agent in addition to ethylenediamine, chelating agents such as EDTA, iminodiacetic acid and utrilotriacetic acid, and various polyamines can be used.
  • the above production method can be introduced on various surfaces in addition to the Balta polymerization.
  • materials that can introduce polymerizable functional groups such as polystyrene resin, silica particles, carbon nanotubes, glass substrates, gold substrates, and various metal substrates.
  • the array chip according to the present invention is not particularly limited to other structures, substrates, etc., as long as the imprinted polymer in the ⁇ 1> column is produced using different proteins in a bowl shape and provided in large numbers.
  • the fixing method on the substrate is not particularly limited, and conventional thin film forming technology and protein array technology can be applied.
  • the imprint polymer preferably has a dye-modifying monomer.
  • Proteins can be easily detected by using dye-modifying monomers such as coumarin 'dansyl' FITC 'anthracene as fluorescent dyes. Possible detection methods include fluorescence spectrum change, fluorescence lifetime measurement, and fluorescence energy transfer.
  • dyes There are three types of dyes: a method in which a monomer having a polymerizable functional group introduced is copolymerized, a method in which it is adsorbed on a polymerized polymer, and a method in which it is covalently bonded to a metal complex and introduced near the recognition site.
  • the protein identification method and purification method according to the present invention should utilize the difference in the adsorption property to the imprint polymer in the column 1> above.
  • noturn analysis can be performed by preparing an array of imprinted polymers prepared with different complexes, cage proteins, metals, and crosslinking ratios.
  • the protein can be identified by using the array chip in the ⁇ 3> column and detecting and analyzing the signal emitted by the dye-modified monomer.
  • deconvolution can also detect multiple proteins quantitatively in cells and tissue forces. It becomes possible.
  • Protein recognition can also be used for refolding. In other words, it is possible to recognize only proteins that have a certain tertiary structure state among specific proteins. Proteins are normally stretched when in a denatured state and are folded in their natural state. By imprinting this natural state, only the protein in the natural state can be bound and protein folding is promoted.
  • a denatured protein and an imprinted polymer may be brought into contact with each other in a regeneration buffer!
  • the regeneration buffer is a buffer solution containing the imprint polymer prepared above.
  • a 10 mg / mL imprint polymer and a Tris buffer solution containing 50 mM NaCl and pH 7.4 can be used.
  • a method of bringing the denatured protein into contact with the imprint polymer there are a batch method in which the imprint polymer is not fixed, and a flow method in which the imprint polymer is fixed in a shape such as a column.
  • the contact method can be appropriately set according to the target protein and other conditions.
  • the Notch method results in a protein-polymer complex, so that after several hours of refolding, the target protein can be recovered by dissociation with salt or pH.
  • the Flow method only the protein that has been folded into the natural state binds strongly, so if it elutes quickly without being folded, re-fold it again.
  • the resulting polymer can inhibit protein aggregation and promote protein folding. it can.
  • a denatured state protein that is, a non-natural state protein is prepared by heat from the outside or a denaturant-added agent. Ribonuclease is completely denatured at 80 ° C to 90 ° C. The above-mentioned metal complex is added to this, and the imprinted polymer specific to the denatured protein is obtained by thermal polymerization. One is possible. The obtained polymer can be used for detection of denatured state and protein removal. Imprinting of proteins denatured with a denaturant is also possible.
  • the imprint polymer production method of the present invention can be used as a method for non-covalent immobilization without degrading protein recognition ability.
  • a target protein is first complexed with a substrate that specifically binds to the protein.
  • a substrate that specifically binds to the protein.
  • the protein can be immobilized with the substrate recognition site blocked.
  • a metal complex having an affinity of about K to 10 3 ⁇ is used so that the metal does not competitively inhibit the protein-substrate interaction.
  • the specific ligand (2'-CMP) can be dissociated by using neutral to weakly basic and high salt conditions.
  • the obtained polymer is immobilized without binding the functional monomer in the vicinity of the substrate, so that it can be used as a sensing or separation material while retaining the substrate recognition ability in the natural state.
  • the imprint polymer according to the present invention has a recognition site to which a target protein binds reversibly, and the recognition site has a metal complex and a cage or a substrate inhibitor.
  • specificity is improved compared to conventional imprinted polymers, proteins can be identified more accurately, and application to sensor arrays enables more accurate identification and analysis of the composition of the mixture. Possible.
  • the method for producing an imprint polymer according to the present invention uses a metal complex that coordinates a metal capable of binding to a target protein, and a substrate or a substrate inhibitor of the target protein as a functional monomer. It is characterized by including a fixing step for fixing the functional monomer.
  • Specific binding by metal coordination is non-specific binding, that is, a protein binding site. It can be made clear by eliminating the coordination ability of a powerful metal complex that cannot be formed successfully. Therefore, after complexing the protein with the metal complex or immobilizing it, the addition of a chelating agent or the like masks the extra metal involved in non-specific binding, and the non-specific binding. Coordination bonds can be reduced. Such precise expression is unique to metal coordination, which is not possible with conventional imprinting methods such as hydrogen bonding, ionic bonding, and! /.
  • Various protein imprinted polymers were synthesized to form an imprinted polymer array, a plurality of proteins were recombined with each other, and the amount of binding of each protein to each polymer was analyzed by principal component analysis. When converted to three-dimensional data, the protein mixture was completely classified into component compositions. This indicates that the protein contained in the imprinted polymer array can be identified for complex samples. It is also possible to classify unknown proteins, which is very useful for screening proteins with useful functions and profiling protein compositions.
  • a protein expresses a function by taking a folded structure. Therefore, by using the molecular recognition ability of the imprinted polymer of the present invention, a protein that has been successfully folded can be stabilized by a complex structure with the folded structure and converted into a correct folded structure.
  • a protein is artificially produced, it is particularly important in the biotechnology field to correctly fold the produced polypeptide and express its function, and the present invention only provides a method for protein identification and separation. In addition, it plays the role of an artificial molecular chaperone that induces protein folding.
  • proteins take a one helix or / 3 sheet as a secondary structure, which greatly affects the overall higher order structure and sometimes causes disease.
  • detection and discrimination of higher-order structures of these proteins has become a major issue.
  • the protein imprinting of the present invention provides a solution, and it is also possible to produce an element for discriminating the folded state of the peptide.
  • a substance capable of recognizing the target protein can be produced by a simpler method than natural compounds such as antibodies.
  • the metal complex functional monomer was synthesized by the following steps.
  • Cyclene tetrahydrochloride 500 mg was dissolved in methylene chloride, cooled to 0 ° C in the presence of 5 equivalents of triethylamine, and then 03.0 equivalents of (Boc) dissolved in methylene chloride was slowly added.
  • ZL'f HD HD ⁇ V ' ⁇ ' 3 ⁇ 4)
  • ZZ 'HD HD ⁇ V' ⁇ '3 ⁇ 4) 9Z "S' (HV ⁇ ⁇ U'9 '(' Hf 0VL-6VL 9 :( ⁇ 3) ⁇ ⁇ - ⁇ ⁇ 'OST
  • Compound 5 was subjected to a condensation reaction by adding 1 equivalent of styrene carboxylic acid, 1 equivalent of WSC, 1 equivalent of HOBt, and 1 equivalent of DIEA in methylene chloride. After washing with citrate and sodium bicarbonate, purification was performed by silica gel chromatography.
  • a 5 mM methanol solution of N, N, -bis (acryloyl) cystamine was prepared, and a gold substrate was immersed in the solution to introduce acrylic acid, which is a polymerizable functional group, onto the substrate.
  • the polymer thin film obtained without the complex with protein by the gold substrate obtained in [42] above is called a non-imprinted polymer. This can be done in the same way. wear.
  • the function as an SPR sensor was compared under certain conditions. The results are shown in FIG. As a result, it was found that the imprinting had higher binding ability to ribonuclease A. Furthermore, by adding the metal again and returning it to the original complex, it was also possible to return the binding amount to the original state again (Fig. 15).
  • Ribonuclease was imprinted using 2,1 UMP styrene synthesized in [3] above as a functional monomer.
  • UMP styrene synthesized in [3] above
  • a binding affinity of about 10 times that of a homogeneous system was obtained by immobilization.
  • the amount of saturated binding and affinity were improved by imprinting.
  • LY lysozyme
  • CY cytochrome C
  • RN ribonuclease A
  • LA lactalbumin
  • two functional polymers copper-ethylenediamine (Cu (en)), copper One cyclen (Cu (cyc) combined with 8 types of imprinted polymers LY-Cu (en) IP, LY-Cu (cyc) IP, CY—Cu (en) IP, CY—Cu (cyc) IP, RN) — Cu (en) IP, RN— Cu (cyc) IP, LA— Cu (en) IP, LA-Cu (cyc) IP, and two non-imprinted polymers (Cu (en) -NIP, Cu (en )-NI P) was produced.
  • imprinted polymers LY-Cu (en) IP, LY-Cu (cyc) IP, CY—Cu (en) IP, CY—Cu (cyc) IP, RN) — Cu (en) IP, RN— Cu (cyc) IP,
  • the head portion of the name of the imprint polymer indicates a cage protein, and Cu (en) described next indicates the functional monomer used.
  • the non-imprinted polymer was produced in the same manner as the imprinted polymer, except that the anchor protein was not added during polymerization.
  • the first half of the name of the non-imprinted polymer refers to the functional polymer used and is distinguished from IP by adding NIP at the end of the name.
  • the specific production was performed according to the recipe shown in Table 1. Dissolve Cu (en) or Cu (cyc) in 15 mL of Tris buffer (TB, 50 mM, pH 7.4) to the specified concentration for each polymer shown in Table 1. After adjusting the pH to 7.4, the solution was diluted to 20 mL (prepolymer mix).
  • the obtained imprinted polymer was lightly crushed with a spatula, washed with 0.5 M NaCl aqueous solution and water, and then washed with 5% acetic acid overnight. Then, it wash
  • the amount of washing of the protein-type saddle protein was determined by measuring the absorbance of the washing solution at 280 nm using a visible ultraviolet spectrophotometer. The washed polymer was lyophilized for 24 hours.
  • HPLC conditions are as follows.
  • HPLC conditions are as follows.
  • Figure 10 shows a chromatogram of protein separation using hydrophobic chromatography.
  • the salt concentration linear gradient allows the four proteins to be completely separated in as little as 8 minutes.
  • Each polymer is considered as one sensor channel, and it is thought to be output from a sensor consisting of 5 channels each. It can be seen that the five data plots of the same protein form one cluster with the same weight. Each cluster was completely independent without overlapping each other. A slightly different cluster distribution was observed depending on the two types of functional monomers used.
  • each polymer exhibits a characteristic response to each protein, and a principal component analysis is performed to analyze each protein.
  • the data was separated into four completely independent clusters.
  • cytochrome C and lactalbumin also formed independent clusters. And it was separated into a total of 6 clusters, and it was shown that the identification of the component in the protein mixture was possible by using this method.
  • the lysozyme imprinted polymer and non-imprinted polymer were synthesized in the same manner as LY-Cu (en) IP and Cu (en) -NIP in [11] above. These two polymers were used in the modified lysozyme regeneration experiment described below.
  • lysozyme Dissolve lysozyme to a concentration of 10.0 mg / mL in denaturing buffer (8M urea, ImM EDTA, 10 mM DTT, 0. ImM Tris—HCl, pH 8.5) and stir at 38 ° C for 2.5 hours Then, denaturation was performed.
  • denaturing buffer 8M urea, ImM EDTA, 10 mM DTT, 0. ImM Tris—HCl, pH 8.5
  • the modified solution is referred to as a modified solution.
  • a lysozyme substrate (Micrococcus lysodeikticus (dried cell wall)) was suspended in 50 mM Tris buffer (pH 6.2) at a concentration of 0.25 mg / mL. 3 mL of the solution was placed in a constant-temperature cell (35 ° C-constant), mixed with lysozyme solution (9 ⁇ L), and the decrease in absorbance at 450 nm was measured using a visible ultraviolet spectrophotometer. The enzyme activity of lysozyme was determined from the slope of the measurement start time (initial rate of enzyme reaction), and the lysozyme solution to be measured was diluted with 50 mM Tris buffer so that the activity was in a range proportional to the concentration.
  • the amount of enzyme that gives a decrease in absorbance of 1 Abs per minute when 3 mL of the suspension and 9 L of lysozyme solution are mixed in the cell is defined as 1 U, and lysozyme Create a calibration curve plotting the relationship between concentration and enzyme activity (Not shown).
  • 3 mL of denaturing solution is mixed with 27 mL of regeneration buffer (1.5 M Urea, 3 mM Glutathione reduced form, 5 mM Glutathione oxidized form, 1 mM EDTA, 0.1 M Tris—HC1, pH 8.0) and diluted 10-fold did. 30 mL each of the diluted solution was prepared in three containers.
  • the absorbance of the solution was measured immediately after dilution, and this was used as the initial concentration.
  • 60 mg each of LY-Cu (en) IP or Cu (cyc) -NIP was added to the remaining two.
  • 0.3 mL of the denaturing solution was poured into solid-phase synthesis tubes filled with 1.5 g of LY-Cu (en) IP or Cu (en) -NIP, respectively, to adsorb lysozyme to these polymers.
  • 2.7 mL of regeneration buffer 1.5 M Urea, 1 mM EDTA, 0.1 M Tris-HC1, pH 8.0
  • the adsorbed protein was eluted with 50 mM citrate-sodium taenoate buffer (pH 6.0), and 3 mL was collected at the first time, and then 15 mL fractions were taken every 2 mL. The absorbance of each fraction was measured, and it was confirmed that an initial amount of approximately 100% was recovered.
  • the enzyme activity of the solution in which all the fractions were combined was measured by the same method as in 1) above.
  • 2.7 mL of a regenerating solution was added dropwise to 0.3 mL of a denatured solution over the same time as the polymer was added, and the enzyme activity was also measured.
  • the enzyme activity was measured by reacting 90 ⁇ L of the total fraction containing lysozyme with 3000 ⁇ L of the substrate solution, and performing the same procedure as described above. The refolding rate of lysozyme was determined from the determined enzyme activity.
  • Figure 13 shows the changes over time in the lysozyme refolding rate when the imprinted polymer was added.
  • the refolding rate after 24 and 27 hours was about 90%, which was almost the same as the 8 hour stage. As a result, the refolding rate is expected to reach its peak at about 8 hours.
  • Fig. 14 shows the relationship (chromatogram) between the elution amount and absorbance in [2-4] 2) above. From Fig. 14, it can be seen that the amount of liquid required for elution of lysozyme is greater for LY-Cu (en) IP than for Cu (en) -NIP. This is thought to be because the imprint polymer has higher lysozyme retention.
  • the refolding rate was high due to the imprint polymer (data not shown), similar to the batch type result in 1) above. That is, LY-Cu (en) IP functioned as a molecular chaperone.
  • -CMP was added in an equivalent amount of pH 5.5 to 1 ribonuclease A 300 M and complexed. To this was added 5 equivalents of a copper-cyclene complex and allowed to associate. To this was added 3 equivalents of acrylamide and 1.5 equivalents of methylenebisacrylamide, and radical initiators potassium persulfate and TEMED were added to perform radical polymerization. After that, the pH was adjusted to 7.4 and 2, — CMP was removed by washing to obtain an immobilized ribonuclease whose substrate binding site was not blocked. It was found that the obtained immobilized protein was immobilized without losing its original activity, compared with the protein immobilized without adding 2'-CMP.
  • the imprinted polymer of the present invention is a separation agent such as chromatography for identification and isolation of proteins in life science, food field, environmental field, biotechnology, diagnostic reagent and protein chip, biotechnology. Identification of proteins artificially produced using technology can be used for refolding processes.

Abstract

Disclosed is an imprint polymer having a recognition site which is reversibly bound to a target protein. Since the recognition site has at least one of a metal complex and a substrate inhibitor, the imprint polymer can have a high specificity for the target protein. Such an imprint polymer is applicable to protein chips or the like.

Description

明 細 書  Specification
インプリントポリマーおよびその利用  Imprint polymer and use thereof
技術分野  Technical field
[0001] 本発明は、標的分子を特異的に認識する分子インプリント技術に関するものであり [0001] The present invention relates to a molecular imprint technique for specifically recognizing a target molecule.
、特に、タンパク質を認識可能なインプリントポリマーの合成法、およびその利用方法 に関するものである。 In particular, the present invention relates to a method for synthesizing an imprinted polymer capable of recognizing a protein, and a method for using the method.
背景技術  Background art
[0002] ポストゲノムにおけるタンパク質の解析とその応用は現在の科学技術における最も 重要なテーマである。その中で、 X線結晶解析等により得られた基礎情報から、タン パク質を医療 ·工業等の様々な分野で応用していくためには、簡便かつ安価な分離' 検出系の構築が非常に重要である。これが可能となれば、医薬を安価で大量に分離 するための機能材料化、ある 、は簡便な診断法として期待されるプロテインチップへ の応用が可能となる。現在、主に開発がなされているのは、抗体や天然のレセプター 、目的のタンパク質に対する特異的なリガンドを固定ィ匕したチップなどである。しかし ながら、これらのリガンドの探索や調製は非常に煩雑で時間を要する作業であり、コ ストも非常に高くなるため、簡便で汎用性の高い人工の分離'検出系の構築が必須 である。すなわち、目的とするタンパク質に対して親和性をもつ非タンパク質 ·非核酸 の人工マテリアルが必要である。  [0002] Protein analysis in the post-genome and its application are the most important themes in current science and technology. In order to apply the protein in various fields such as medicine and industry based on the basic information obtained by X-ray crystallography etc., the construction of a simple and inexpensive separation and detection system is very important. Is important to. If this becomes possible, it will be possible to make functional materials for separating pharmaceuticals in large quantities at low cost, or to apply to protein chips that are expected as simple diagnostic methods. Currently developed mainly are antibodies, natural receptors, and chips with specific ligands for the protein of interest. However, the search and preparation of these ligands is very complicated and time consuming, and the cost is very high. Therefore, it is essential to construct a simple and versatile artificial separation and detection system. In other words, a non-protein / non-nucleic acid artificial material having an affinity for the target protein is required.
[0003] 以上のような特定のタンパク質、すなわちポリペプチドと相互作用する分離 ·検出系 が構築 '確立されれば、タンパク質の検出や分離だけでなぐあるタンパク質の高次 構造を認識'識別したり、特定の高次構造を安定化させるマテリアルの調製が可能と なる。また、タンパク質を非共有結合的に固定ィ匕する方法としても、重要な技術となる  [0003] When a separation / detection system that interacts with a specific protein, that is, a polypeptide as described above, has been established, it can recognize and identify the higher-order structure of a protein that can only be detected and separated. This makes it possible to prepare a material that stabilizes a specific higher-order structure. It is also an important technique for immobilizing proteins non-covalently.
[0004] タンパク質を構成するアミノ酸には、金属配位しやすい残基がいくつか含まれてい る。ヒスチジン、トリブトファン、システィンといった残基は非共有電子対をもつアミノ酸 残基であり、銅'ニッケルなどの遷移金属などに対して効率的な配位結合を形成する 。そこで、これらの遷移金属、及びキレート効果により強固に金属を捕捉する配位子 の組み合わせによりポリペプチドを認識すればよい。 [0004] Amino acids constituting proteins include several residues that are easily metal-coordinated. Residues such as histidine, tributophan, and cysteine are amino acid residues having an unshared electron pair, and form efficient coordination bonds to transition metals such as copper and nickel. Therefore, these transition metals and ligands that capture metals firmly by the chelate effect A polypeptide may be recognized by the combination of
[0005] 標的分子を認識する技術として、分子インプリント法と呼ばれる技術が注目されて いる。分子インプリンティングとは、標的分子と相補的な認識部位を有する高分子を 得る方法である。詳細は以下の通りである。まず、標的分子を铸型分子とし、铸型分 子と、この铸型分子と結合可能な機能性モノマーとの複合体を形成させる。そして得 られた複合体を架橋剤と共に重合させ、架橋高分子を得る。その後、铸型分子を溶 媒等により除去することにより、高分子内に、標的分子に対して相補的な認識部位を 構築する。  [0005] As a technique for recognizing a target molecule, a technique called a molecular imprint method has attracted attention. Molecular imprinting is a method for obtaining a polymer having a recognition site complementary to a target molecule. Details are as follows. First, the target molecule is a cage molecule, and a complex of the cage molecule and a functional monomer that can bind to the cage molecule is formed. Then, the obtained composite is polymerized with a crosslinking agent to obtain a crosslinked polymer. After that, by removing the cage molecule with a solvent or the like, a recognition site complementary to the target molecule is constructed in the polymer.
[0006] 分子インプリンティングの技術はここ数十年にわたり開発され、この技術によって得 られた高分子は、吸着剤や高速液体クロマトグラフィーの分離担体としての利用が検 討されている。  [0006] The technology of molecular imprinting has been developed over the last few decades, and the use of the polymer obtained by this technology as an adsorbent or a separation carrier for high performance liquid chromatography is being investigated.
[0007] 従来行われてきた分子インプリント技術は、主に低分子化合物に対して機能するも のである。これまで、タンパク質などのように巨大な分子に対して機能した例は少ない 力 次のような例がある。  [0007] The molecular imprint technique that has been conventionally performed mainly functions for low molecular weight compounds. So far, there are few examples that have worked on large molecules such as proteins.
[0008] Mosbachらは、金属キレートモノマー N- (4- vinyl)- benzyl iminodiacetic acidと Cu(II )との配位結合を利用したリボヌクレアーゼ Aのインプリンティングを報告している（非 特許文献 1 : M. Kempe, M. Glad, and K. Mosbach, J. Molec. Recogn. 1995, 8, 3539 ) o具体的には、シリカゲル表面上で、上記金属キレートモノマー、 Cu(II)、およびリボ ヌクレアーゼ Aの複合体を形成させたところに架橋剤を加えることで、金属キレートモ ノマ一間を重合させている。  [0008] Mosbach et al. Reported imprinting of ribonuclease A using a coordinate bond between metal chelate monomer N- (4-vinyl) -benzyl iminodiacetic acid and Cu (II) (Non-patent Document 1: M. Kempe, M. Glad, and K. Mosbach, J. Molec. Recogn. 1995, 8, 3539) o Specifically, on the silica gel surface, the metal chelate monomer, Cu (II), and ribonuclease A The metal chelate monomer is polymerized by adding a cross-linking agent to the complex.
[0009] また、 Minouraらは、同じくシリカゲルをサポートとして用いてグルコースォキシダー ゼのインプリンティングを行った（非特許文献 2 : M. Burow and N. Minoura, Biochem. Biophys. Res. Commun. 1996, 227, 419-422)。正電荷を有する 2- (ジメチルァミノ)ェ チルメタタリレート、負電荷を有するアクリル酸、およびグルコースォキシダーゼの基 質であるグルコースを側鎖に有するダルコシルォキシェチルメタタリレート、電荷を持 たな 、アクリルアミドと 4種類の機能性モノマーを用い、主に 3種類の相互作用で铸 型分子の認識を行うインプリントポリマーを合成している。  [0009] Further, Minoura et al. Also imprinted glucose oxidase using silica gel as a support (Non-patent Document 2: M. Burow and N. Minoura, Biochem. Biophys. Res. Commun. 1996). , 227, 419-422). Positively charged 2- (dimethylamino) ethyl metatalylate, negatively charged acrylic acid, and glucosidase base glucosyl glucosylase in the side chain, charged In addition, acrylamide and four types of functional monomers are used to synthesize imprinted polymer that recognizes vertical molecules mainly through three types of interactions.
[0010] し力しながら、これらのインプリンティング技術では、プロテインチップ ·プロテインァ レイに応用可能な高い基質特異性を有する高分子を得ることは困難である。すなわ ち、上記従来の方法で製造されたインプリントポリマーでは、結合選択性や、センサ 一、センサーアレイ、センサーチップとしての利用可能性の点で問題があった。タン ノ ク質検出素子、分離材料を創製するためには、高い基質選択性を与えるインプリ ンティング技術が必要となる。 [0010] However, with these imprinting technologies, protein chips It is difficult to obtain a polymer having high substrate specificity applicable to ray. That is, the imprint polymer produced by the conventional method has problems in terms of bond selectivity and applicability as a sensor, sensor array, or sensor chip. In order to create a protein detection element and a separation material, an implementation technology that provides high substrate selectivity is required.
[0011] 本発明は、上記従来の問題に鑑みたものであり、その目的は、より結合特異性の高 いインプリントポリマーを提供すると共に、色素修飾によるインプリントポリマーセンサ 一、その製造方法、およびその代表的な利用方法であるマイクロアレイ等を提供する ことにある。また、従来のインプリンティングの欠点であった低い選択性を克服するた め、金属錯体特有の多価配位による特異性の向上、及び非特異的相互作用の抑制 することを課題とする。  [0011] The present invention has been made in view of the above-described conventional problems, and an object of the present invention is to provide an imprint polymer with higher binding specificity and to provide an imprint polymer sensor by dye modification. Another object of the present invention is to provide a microarray that is a typical method of use. In addition, in order to overcome the low selectivity that has been a drawback of conventional imprinting, it is an object to improve specificity and suppress non-specific interactions due to the multivalent coordination unique to metal complexes.
発明の開示  Disclosure of the invention
[0012] 認識部位に金属錯体および基質阻害剤の少なくとも一方を有するインプリントポリ マーは、従来のインプリントポリマーよりも高い選択性を有することを見出し、本発明 を完成させるに至った。本発明は、上記従来の課題に鑑みたものであり、以下の発 明を含有する。  [0012] An imprint polymer having at least one of a metal complex and a substrate inhibitor at a recognition site has been found to have higher selectivity than a conventional imprint polymer, and the present invention has been completed. The present invention has been made in view of the above-described conventional problems, and includes the following inventions.
[0013] (1)標的タンパク質に可逆的に結合する認識部位を有し、上記認識部位は、金属 錯体および基質阻害剤の少なくとも一方を有することを特徴とするインプリントポリマ  [0013] (1) An imprint polymer comprising a recognition site that reversibly binds to a target protein, wherein the recognition site has at least one of a metal complex and a substrate inhibitor.
[0014] (2)上記（1)のインプリントポリマーであって、上記金属錯体は、標的分子と結合す ることによって結合シグナルを発することを特徴とするインプリントポリマー。 [0014] (2) The imprint polymer according to (1), wherein the metal complex emits a binding signal by binding to a target molecule.
[0015] (3)上記（1)または（2)のインプリントポリマーを備えるアレイチップ。 [0015] (3) An array chip comprising the imprint polymer of (1) or (2).
[0016] (4)上記（1)または（2)のインプリントポリマーに対する吸着特性の違いからタンパ ク質を同定する方法。 [0016] (4) A method for identifying a protein from the difference in adsorption characteristics with respect to the imprint polymer of (1) or (2) above.
[0017] (5)上記（1)または（2)のインプリントポリマーに対する吸着特性の違いからタンパ ク質を精製する方法。  [0017] (5) A method for purifying a protein from the difference in adsorption characteristics with respect to the imprint polymer of (1) or (2).
[0018] (6)上記（1)または（2)のインプリントポリマーによってタンパク質をリフォールディン グする方法。 [0019] (7)上記（1)または（2)のインプリントポリマーによってタンパク質の折りたたみ状態 を判別する方法。 [0018] (6) A method of refolding a protein with the imprint polymer of (1) or (2) above. [0019] (7) A method for discriminating a protein folding state by the imprint polymer of (1) or (2).
[0020] (8)標的タンパク質と機能性モノマーとの複合体を形成する複合体形成工程と、上 記複合体中の機能性モノマーを重合させる重合工程とを含み、上記機能性モノマー は、標的タンパク質と結合可能な金属に配位する配位子、および、標的タンパク質の 基質阻害剤の少なくとも一方を含むことを特徴とするインプリントポリマーの製造方法  [0020] (8) comprising a complex forming step of forming a complex of the target protein and the functional monomer, and a polymerization step of polymerizing the functional monomer in the complex, wherein the functional monomer is a target A method for producing an imprinted polymer comprising a ligand coordinated to a metal capable of binding to a protein and a substrate inhibitor of a target protein
[0021] (9)上記（8)のインプリントポリマーの製造方法であって、上記複合体形成工程によ つて形成された複合体を単離する単離工程を含み、当該単離工程後に重合工程を 行うことを特徴とするインプリントポリマーの製造方法。 [0021] (9) The method for producing an imprint polymer of (8) above, comprising an isolation step of isolating the complex formed by the complex formation step, and polymerization after the isolation step A process for producing an imprint polymer, characterized by comprising the steps of:
[0022] (10)上記（8)または（9)に記載のインプリントポリマーの製造方法であって、上記 複合体形成工程および重合工程の少なくとも一方を行った後に、上記金属に配位可 能な配位子によって、タンパク質と相補的な位置力も外れた金属をブロックすることを 特徴とするインプリントポリマーの製造方法。  [0022] (10) The method for producing an imprint polymer according to (8) or (9) above, wherein the imprint polymer can be coordinated to the metal after performing at least one of the complex formation step and the polymerization step. A method for producing an imprinted polymer, characterized in that a metal having a complementary positional force to a protein is blocked by a simple ligand.
[0023] (11)上記（8)〜（10)のいずれかのインプリントポリマーの製造方法であって、上記 重合工程を行った後、キレート剤を添加すること、又は pHを変化させることによって 上記配位子力 金属を脱離させ、その後、上記金属とは異なる金属を配位させること によって、上記インプリントポリマーの機能改変を行う工程を含むことを特徴とするィ ンプリントポリマーの製造方法。  [0023] (11) A method for producing an imprinted polymer according to any one of (8) to (10) above, wherein after the polymerization step is performed, a chelating agent is added or the pH is changed. The method for producing an imprint polymer comprising the step of modifying the function of the imprint polymer by detaching the ligand force metal and then coordinating a metal different from the metal. .
[0024] (12)標的タンパク質と当該標的タンパク質の基質とを複合化させた基質一タンパク 質複合体と、機能性モノマーとを複合化させる工程と、上記工程後に機能性モノマー を重合させる工程を行うことを特徴とするタンパク質の固定ィ匕方法。  [0024] (12) A step of complexing a substrate-protein complex obtained by complexing a target protein and a substrate of the target protein and a functional monomer, and a step of polymerizing the functional monomer after the above step A protein immobilization method characterized by comprising:
[0025] (13)上記（12)に記載のタンパク質の固定ィ匕方法であって、上記機能性モノマー は、上記標的タンパク質と結合可能な金属に配位する配位子を含むことを特徴とす るタンパク質の固定化方法。  [0025] (13) The protein immobilization method according to (12) above, wherein the functional monomer includes a ligand that coordinates to a metal that can bind to the target protein. A method for immobilizing proteins.
[0026] 本発明のさらに他の目的、特徴、および優れた点は、以下に示す記載によって十 分わ力るであろう。また、本発明の利益は、添付図面を参照した次の説明で明白にな るであろう。 図面の簡単な説明 [0026] Still other objects, features, and advantages of the present invention will be sufficiently enhanced by the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings. Brief Description of Drawings
[図 1]本発明におけるインプリントポリマーの製造方法を示す図面である。 FIG. 1 is a drawing showing a method for producing an imprint polymer in the present invention.
[図 2]本発明の実施の形態に係る錯体の構造式である。 FIG. 2 is a structural formula of a complex according to an embodiment of the present invention.
[図 3]本発明の実施の形態に係る色素修飾モノマーの構造式を示す図面であり、 (a) はスチレン修飾モノマー、 （b)はサイクレン修飾モノマーを示す。  FIG. 3 is a drawing showing a structural formula of a dye-modified monomer according to an embodiment of the present invention, where (a) shows a styrene-modified monomer and (b) shows a cyclen-modified monomer.
[図 4]本発明の実施の形態に係る色素修飾錯体ースチレンモノマーの共有結合体 (ト ル)の構造式を示す図面であり、（a)はエチレンジァミン一アントラセン、 （b)はサイク レン一アントラセン、 （c)はサイクレン一ダンシル、 (d)はエチレンジァミン一ダンシノレ 修飾体である。 FIG. 4 is a drawing showing the structural formula of a covalent bond (tol) of a dye-modified complex-styrene monomer according to an embodiment of the present invention, where (a) is ethylenediamine-anthracene, and (b) is cyclen. Anthracene, (c) is a cyclen-dansyl, and (d) is an ethylenediamine-dansinore modified.
[図 5]本発明の実施の形態に係るスチレン修飾リボヌクレアーゼ阻害剤の構造式を示 す図面である。  FIG. 5 is a drawing showing a structural formula of a styrene-modified ribonuclease inhibitor according to an embodiment of the present invention.
[図 6]本発明の実施例に係るサイクレン修飾体の合成スキームを示す図面である。  FIG. 6 is a drawing showing a synthesis scheme of a modified cyclone according to an example of the present invention.
[図 7]本発明の実施例に係るエチレンジァミン修飾体の合成スキームを示す図面であ る。 FIG. 7 is a drawing showing a synthesis scheme of modified ethylenediamine according to an example of the present invention.
[図 8]本発明の実施例に係るスチレン修飾リン酸ィ匕 UMPの合成スキームを示す図面 である。  FIG. 8 is a drawing showing a synthesis scheme of styrene-modified phosphate UMP according to an example of the present invention.
[図 9]本発明の実施例に係るインプリントポリマーおよび非インプリントポリマーのリボ ヌクレアーゼ Aに対するセンサーグラムである。  FIG. 9 is a sensorgram for ribonuclease A of imprinted polymer and non-imprinted polymer according to an example of the present invention.
[図 10]本発明の実施例における疎水性クロマトグラフィーを用いたタンパク質分離の クロマトグラムである。  FIG. 10 is a chromatogram of protein separation using hydrophobic chromatography in an example of the present invention.
[図 ll]Cu(en)を用いて調製した 5種類のポリマーに対するミオグロビン（Al)、リボヌク レアーゼ A (A2)、リゾチーム（A3)、アルブミン (A4)、シトクロム C (A5)、ラクトアルブミン [Figure ll] Myoglobin (Al), ribonuclease A (A2), lysozyme (A3), albumin (A4), cytochrome C (A5), lactalbumin for five types of polymers prepared using Cu (en)
(A6)の吸着挙動を主成分分析した結果を示すデータプロットである。 It is a data plot which shows the result of having carried out the principal component analysis of the adsorption behavior of (A6).
[図 12]Cu(cyc)を用いて調製した 5種類のポリマーに対するミオグロビン（Al)、リボヌク レアーゼ A (A2)、リゾチーム（A3)、アルブミン (A4)、シトクロム C (A5)、ラクトアルブミン[Fig. 12] Myoglobin (Al), ribonuclease A (A2), lysozyme (A3), albumin (A4), cytochrome C (A5), lactalbumin for five polymers prepared using Cu (cyc)
(A6)の吸着挙動を主成分分析した結果を示すデータプロットである。 It is a data plot which shows the result of having carried out the principal component analysis of the adsorption behavior of (A6).
[図 13]LY-Cu(en)IP、 Cu(en)- MIP無添カ卩および添カ卩時におけるリフォールデイング率 の経時変化を示す図面である。 [図 14]flow型の再活性ィ匕方法におけるリゾチーム溶出量の経時変化を示すグラフで ある。 FIG. 13 is a drawing showing time-dependent changes in the refolding rate when LY-Cu (en) IP and Cu (en) -MIP are not added and when they are added. FIG. 14 is a graph showing the change over time in the elution amount of lysozyme in the flow type reactivation method.
[図 15]EDTAを添加して結合親和性を低下させた後、さらに銅イオンを添加して活性 を回復させたセンサーグラムである。  FIG. 15 is a sensorgram in which the binding affinity is lowered by adding EDTA and then the activity is restored by adding copper ion.
[図 16]ビュルィ匕したエチレンジァミンを機能性モノマーとして合成したインプリントポリ マーについて、各タンパク質に対するブランクポリマーとの相対親和性を示したダラ フである。  [Fig. 16] Draft showing relative affinities of imprinted polymers synthesized with functional ethylene diamine as a functional monomer for each protein.
[図 17]ビュルィ匕したサイクレンを機能性モノマーとして合成したインプリントポリマーに っ 、て、各タンパク質に対するブランクポリマーとの相対親和性を示したグラフである  [Fig. 17] A graph showing the relative affinity of each imprinted polymer synthesized from bulky cyclen as a functional monomer with a blank polymer.
[図 18]インプリントポリマー、ブランクポリマー、 GEMAのみ、の三者のポリマーについ てのリボヌクレアーゼ Aに対する親和性の塩濃度依存性である。 [FIG. 18] The affinity of ribonuclease A for the three polymers, imprint polymer, blank polymer, and GEMA alone, is dependent on the salt concentration.
[図 19]リボヌクレアーゼ Aを固定化した基板に対する Zn(TCPP(Tetrakis(4- carboxyph enyl)porphine》の結合を SPRセンサーグラムで解析した例である。  FIG. 19 shows an example of analyzing the binding of Zn (TCPP (Tetrakis (4-carboxyphenyl) porphine)) to a substrate on which ribonuclease A is immobilized using an SPR sensorgram.
[図 20]リボヌクレアーゼ Aに対して Zn[TCPP] (Tetrakis(4- carboxyphenyl)porphine)を 滴定した時の UV吸収スペクトルである。  FIG. 20 is a UV absorption spectrum when titrating Zn [TCPP] (Tetrakis (4-carboxyphenyl) porphine) against ribonuclease A.
[図 21]ビュルィ匕した Zn[TCPP]を機能性モノマーとして合成したインプリントポリマーに っ 、て、各タンパク質に対するブランクポリマーとの相対親和性を示したグラフである 発明を実施するための最良の形態  FIG. 21 is a graph showing the relative affinity of each of the imprinted polymers synthesized using bulky Zn [TCPP] as a functional monomer and the blank polymer for each protein. BEST MODE FOR CARRYING OUT THE INVENTION Form
[0028] < 1.タンパク質をターゲットとするインプリントポリマー > [0028] <1. Imprint polymer targeting protein>
本発明に係るインプリントポリマーは、標的タンパク質に可逆的に結合する認識部 位を有し、上記認識部位は、金属錯体および基質阻害剤の少なくとも一方を有する 。なお、本明細書における文言「可逆的に結合する」は、共有結合以外の結合を意 味する。また、金属錯体および基質阻害剤は、標的タンパク質によって適宜変更する ことができ、特に限定されるものではない。  The imprint polymer according to the present invention has a recognition site that reversibly binds to a target protein, and the recognition site has at least one of a metal complex and a substrate inhibitor. Note that the phrase “reversibly binds” in this specification means a bond other than a covalent bond. In addition, the metal complex and the substrate inhibitor can be appropriately changed depending on the target protein, and are not particularly limited.
[0029] (1 1)機能性モノマー [0029] (1 1) Functional monomer
インプリントポリマーの組成は特に限定されるものではないが、例えばいわゆる機能 性モノマーが重合してなるポリマーを主成分として含むものが挙げられる。 The composition of the imprint polymer is not particularly limited. And those containing, as a main component, a polymer obtained by polymerizing a functional monomer.
[0030] 機能性モノマーとしては、アクリルアミド、アクリル酸、ダルコシルォキシェチルメタク リレート、 1-ビュルイミダゾール、 4-ビュルピリジン、 2- (ジメチルァミノ)ェチルメタクリレ ート、 2-ヒドロキシェチルメタタリレート等、水溶性のモノマーが好ましい。水溶性のモ ノマーが好ましいのは、生体内物質であるタンパク質は、 pHおよび有機溶媒に対す る敏感性が高ぐ緩衝液中でインプリンティングを行うことが好ま ヽためである。  [0030] Examples of functional monomers include acrylamide, acrylic acid, darcosyloxychetyl methacrylate, 1-butyrimidazole, 4-butyrpyridine, 2- (dimethylamino) ethyl methacrylate, and 2-hydroxyethyl methacrylate. Water-soluble monomers such as rate are preferred. Water-soluble monomers are preferred because proteins, which are in vivo substances, are preferably imprinted in buffers that are highly sensitive to pH and organic solvents.
[0031] また水酸基、アミノ基、カルボキシル基等の水素結合性のモノマーは、標的タンパク 質との間に水素結合を形成することができる。この水素結合によって、補助的に金属 配位による相互作用を補強することができる。また、カルボン酸、 4級アンモ-ゥム、ス ルホン酸、リン酸等はイオン結合による相互作用の補強を与えることができる。  [0031] Hydrogen-bonding monomers such as a hydroxyl group, an amino group, and a carboxyl group can form a hydrogen bond with a target protein. This hydrogen bond can supplement the interaction by metal coordination. Carboxylic acid, quaternary ammonia, sulfonic acid, phosphoric acid, and the like can reinforce interaction by ionic bonds.
[0032] また、機能性モノマーとして、下記（1— 3)の金属錯体および（1—4)の基質阻害剤 の少なくとも一方を含む。詳細には後述する。  [0032] Further, the functional monomer contains at least one of the following metal complex (1-3) and a substrate inhibitor (1-4). Details will be described later.
[0033] (1 2)色素修飾モノマー  [0033] (1 2) Dye-modifying monomer
本発明のインプリントポリマーは、タンパク質の検出のため、色素修飾モノマーを含 んでもよい。具体的な機能性色素としては、メチルオレンジ、メチルレッドなどのァゾ ベンゼン系、ナフトキノン、アントラキノンなどのキノン系、フエノキサジン、マラカイトグ リーンなどのジァリールメタン系およびトリアリールメタン系、フルォレセイン、ローダミ ン Bなどのフルオラン系、インドシァニン、へミシァニンなどのシァニン系、テトラフエ二 ルポルフィリン、フタロシア-ンなどのポルフィリン'フタロシア-ン系、ォキサゾリルェ チリデンイソプロピリデン無水コハク酸、ァリールフルギン酸無水物などのフルギド系 、インジゴ、チ才インジゴなどのインジゴ系、アントラセン、ピレンなどの縮合環系、テト ラチアフルバレンーテトラシァノキノジメタンなどの電荷移動分子系、メタルジチォレ ン、キノリノール金属錯体などの金属錯体系、スチルベン、スチルバゾールなどのス チリル系、スピロべンゾピラン、スピロォキサジンなどのスピロピラン系、ジフリルェテン 、ジチェニノレエテンなどのジァリーノレェテン系、スクァリリウム、クロコニゥムなどのスク ァリリウム系、ケルセチン、アントシァニンなどのフラボノイド系の機能性色素などがあ る。  The imprint polymer of the present invention may contain a dye-modified monomer for protein detection. Specific functional dyes include azobenzenes such as methyl orange and methyl red, quinones such as naphthoquinone and anthraquinone, dialelemethanes such as phenoxazine and malachite green, triarylmethanes, fluorescein, rhodamine B, etc. Fluoranes, indocyanine, hemisyanine and other cyanines, tetraphenylporphyrin, phthalocyanine and other porphyrins' phthalocyanine, oxazolyl tylidene isopropylidene succinic anhydride, fulgide such as allylfulginic anhydride, indigo, Gold, such as indigo such as indigo, condensed ring systems such as anthracene and pyrene, charge transfer molecular systems such as tetrathiafulvalene-tetracyanoquinodimethane, metal dithiolene, quinolinol metal complexes, etc. Genus complex, styryl such as stilbene and stilbazole, spiropyran such as spirobenzopyran and spiroxazine, diarylethenic such as difurylethene and diceninoreethene, squarylium such as squarylium and croconium, quercetin and anthocyanin And other flavonoid functional pigments.
[0034] 特に、クマリン ·ダンシル 'FITC 'アントラセンなどの蛍光色素を用いることが好ましい 。これにより、容易にタンパク質を検出することができる。検出方法としては、蛍光スぺ タトル変化 ·蛍光寿命測定 ·蛍光エネルギー移動が可能である。 [0034] In particular, it is preferable to use a fluorescent dye such as coumarin dansyl 'FITC' anthracene. . Thereby, protein can be detected easily. Possible detection methods include fluorescence spectrum change, fluorescence lifetime measurement, and fluorescence energy transfer.
[0035] 色素は、単に重合性官能基を導入したモノマーを共重合させる方法、重合した高 分子に吸着させる方法、下記（1 3)の金属錯体と共有結合させて認識部位の近傍 に導入する方法の 3通りがある。  [0035] The dye is introduced in the vicinity of a recognition site by simply copolymerizing a monomer having a polymerizable functional group introduced thereto, by adsorbing it to a polymerized high molecule, or by covalently bonding to the metal complex of (13) below. There are three ways.
[0036] 図 3に、本発明に利用可能な色素修飾モノマーの例を示す。また、図 4に、本発明 に利用可能な、色素修飾錯体ースチレンモノマーの共有結合体の構造式を示す。こ れら色素修飾モノマーは、蛍光波長ピーク、及び蛍光寿命の変化によりタンパク質の 結合を検出することができる。色素、及び金属錯体の構造は図に示したものに限定さ れず、上記に示したものおよび下記（1 3)で示すものが利用できる。  FIG. 3 shows an example of a dye-modifying monomer that can be used in the present invention. FIG. 4 shows the structural formula of a covalently bonded complex of a dye-modified complex and a styrene monomer that can be used in the present invention. These dye-modified monomers can detect protein binding based on changes in fluorescence wavelength peak and fluorescence lifetime. The structures of the dye and the metal complex are not limited to those shown in the figure, and those shown above and those shown in the following (13) can be used.
[0037] (1 3)金属錯体  [0037] (1 3) Metal complex
タンパク質を構成するアミノ酸には、金属配位しやす ヽ残基が ヽくつか含まれて ヽ る。ヒスチジン、トリブトファン、システィンといった残基は非共有電子対をもつアミノ酸 残基であり、銅'ニッケルなどの遷移金属などに対して効率的な配位結合を形成する 。本発明のインプリントポリマーは、その認識部位に、これら金属原子を含む金属錯 体を有する。金属錯体は、金属と、その金属に配位する配位子とからなる。本発明に 係るインプリントポリマーの認識部位は、この金属錯体を有するので、金属原子を介 してこれらアミノ酸残基を認識し、標的タンパク質を認識することができる。なお、金属 錯体とは、配位子と、当該配位子に配位結合する金属とからなるが、本実施の形態 においては、「金属錯体」なる文言が、配位子単独である状態 (すなわち金属と配位 結合していない状態）、および金属を配位した状態の、両方を指すことがある。  The amino acids that make up a protein contain a number of や す residues that facilitate metal coordination. Residues such as histidine, tributophan, and cysteine are amino acid residues having an unshared electron pair, and form efficient coordination bonds to transition metals such as copper and nickel. The imprint polymer of the present invention has a metal complex containing these metal atoms at the recognition site. A metal complex consists of a metal and a ligand that coordinates to the metal. Since the recognition site of the imprinted polymer according to the present invention has this metal complex, it can recognize these amino acid residues via the metal atom and recognize the target protein. Note that a metal complex is composed of a ligand and a metal that is coordinated to the ligand, but in this embodiment, the term “metal complex” is a state in which the ligand is alone ( That is, it may refer to both a state in which the metal is not coordinated and a state in which the metal is coordinated.
[0038] 本発明に係るインプリントポリマーは、このように金属原子を介して標的分子を認識 するので、より多様な条件 '範囲で標的タンパク質を認識することができる。例えば、リ ボヌクレアーゼとその代表的なリボヌクレアーゼ阻害剤（2， -CMP)とは、 5. 5付近の 低 PHでなければ高い親和性を得られない。一方、本発明のインプリントポリマーは、 比較的広い範囲の pHでターゲットタンパク質と結合可能であり、他のタンパク質—リ ガンド相互作用では機能しな 、条件にぉ ヽても親和性を獲得することができる。 [0038] Since the imprint polymer according to the present invention recognizes a target molecule through a metal atom in this way, it can recognize a target protein under a wider range of conditions. For example, Li ribonuclease and its typical ribonuclease inhibitor (2, -CMP) and can not obtain a low P H unless high affinity around 5.5. On the other hand, the imprint polymer of the present invention can bind to a target protein in a relatively wide range of pH, does not function in other protein-ligand interactions, and acquires affinity even under conditions. Can do.
[0039] pHだけでなぐ本発明により得られる人工レセプターは天然の抗体等と比較して、 温度や塩濃度においても幅広い範囲で結合可能である。 2'—CMPとリボヌクレア一 ゼとの相互作用においても、高い塩濃度では結合しないことが報告されている力 本 発明のインプリントポリマーはこのような条件でも結合能がある。こうした点においても 、金属錯体を使った認識部位の構築は天然にない長所を有しているといえる。また、 材料化の際に懸念される安定性にお！、ても、ぺプチダーゼによる切断や高次構造変 ィ匕を引き起こす天然のタンパク質 ·抗体と異なり、安定した性能が長期間にわたつて 保持できる。 [0039] Compared with natural antibodies and the like, the artificial receptor obtained by the present invention that is based only on pH, Binding is possible in a wide range of temperature and salt concentration. In the interaction between 2'-CMP and ribonuclease, it has been reported that it does not bind at a high salt concentration. The imprinted polymer of the present invention is capable of binding even under such conditions. In this respect, it can be said that the construction of a recognition site using a metal complex has an advantage not found in nature. In addition, stability is a concern during materialization! However, unlike natural proteins and antibodies that cause peptidase cleavage and conformational changes, stable performance can be maintained over a long period of time.
[0040] 金属配位は、その金属と配位子の組み合わせにおいて多様な選択が可能である。  [0040] The metal coordination can be selected from various combinations of the metal and the ligand.
同じ配位子で金属を脱離させたり、再結合させたりすることも可能であり、機能を自由 に変えることができる。金属は EDTAにより解離させることができ、さらに別の金属を導 入することもできる。同属の配位形式をもつ金属を用いてより親和性を上げたり、異な る配位形態をもつ金属を添加して基質選択性を変化させたりすることも可能である。 また、一部のランタ-ド金属にはペプチド結合の切断活性があり、これを用いて標的 タンパク質を切断することも可能となる。  It is possible to desorb or recombine metals with the same ligand, and the function can be freely changed. The metal can be dissociated by EDTA, and another metal can be introduced. It is also possible to increase the affinity by using a metal having a coordinating form of the same genus or to change the substrate selectivity by adding a metal having a different coordinating form. Some lanthanide metals have peptide bond cleavage activity, which can be used to cleave target proteins.
[0041] また、配位子と配位する金属としては、遷移金属の銅.亜鉛.マンガン.ニッケル.鉄 などの他、希土類金属、蛍光発光するランタニド金属等を利用することができる。  [0041] In addition to transition metals such as copper, zinc, manganese, nickel, and iron, rare earth metals, lanthanide metals that emit fluorescence, and the like can be used as the metal that coordinates with the ligand.
[0042] 配位子には、エチレンジァミン'サイクレン'ジピコリルアミン 'イミノジ酢酸'ユトリロ三 酢酸 · EDTA ·ボルフイリン等の多価配位型錯体が利用できる。  [0042] As the ligand, a polyvalent coordination complex such as ethylenediamine, cyclene, dipicolylamine, iminodiacetic acid, utilotriacetic acid, EDTA, and volphiline can be used.
[0043] 図 2に、本発明に適用可能な金属錯体の一例を示す。  FIG. 2 shows an example of a metal complex applicable to the present invention.
[0044] なお、機能性モノマーは、本欄で述べた配位子および下記（1 4)欄の基質阻害 剤自体であってもよいし、配位子および基質阻害剤における標的タンパク質を認識 する部位とは異なる位置に重合官能基が導入された化合物であってもよ!/、。配位子 および基質阻害剤は、この重合官能基を介して重合することで、インプリントポリマー を形成することができる。なお、本発明においては、重合官能基が導入されていても されていなくても、配位子、基質阻害剤と称するものとする。  [0044] The functional monomer may be the ligand described in this column and the substrate inhibitor itself in the following (14) column, or recognizes the target protein in the ligand and the substrate inhibitor. It may be a compound with a polymerization functional group introduced at a position different from the site! /. Ligand and substrate inhibitor can be polymerized via this polymerization functional group to form an imprint polymer. In the present invention, whether or not a polymerization functional group is introduced is referred to as a ligand or a substrate inhibitor.
[0045] 重合官能基の導入においては、例えば有機合成の手法により、配位子および基質 阻害剤にスチレン又はアクリル酸を導入してもよい。  In introducing the polymerization functional group, styrene or acrylic acid may be introduced into the ligand and the substrate inhibitor by, for example, an organic synthesis method.
[0046] また、錯体同士を結合させて複核錯体を機能性モノマーとすることもできる。 [0047] 金属錯体を使った方法では、タンパク質に特異的に結合できないように固定化され た金属錯体部位を、キレート剤を用いて不活化させることにより、非特異的な結合を 抑制することができる。これにより、タンパク質と特異的に相互作用して固定化された 金属錯体のみが、再結合の際に関与することになり、高い基質選択性が発現される ことになる。 [0046] In addition, the complex can be bonded to each other to form a binuclear complex as a functional monomer. [0047] In the method using a metal complex, nonspecific binding can be suppressed by inactivating a metal complex site immobilized so that it cannot specifically bind to a protein using a chelating agent. it can. As a result, only the metal complex that is immobilized by specifically interacting with the protein is involved in the recombination, and high substrate selectivity is expressed.
[0048] (1 4)基質阻害剤  [0048] (1 4) Substrate inhibitor
本発明において、基質阻害剤とは、標的タンパク質である酵素の基質と同様に、少 なくともその一部が標的タンパク質の基質結合部位に特異的に結合可能な構造を有 する化合物である。ただし、基質阻害剤は、基質とは異なり酵素の触媒作用を受けな いか、または触媒作用を受けてもその反応速度が非常に遅いことが必要である。この ような物質としては、標的タンパク質の基質と類似の構造を有する競合阻害剤、およ び擬基質等が含まれる。これら基質阻害剤は、標的タンパク質との結合の特異性が 高い。そのため、インプリントポリマーに、高い標的認識特性を付与することができる。 また、触媒作用を受けないか、反応速度が非常に遅いので、標的タンパク質の基質 認識部位との結合が安定である。  In the present invention, the substrate inhibitor is a compound having a structure capable of specifically binding at least a part to the substrate binding site of the target protein, like the substrate of the enzyme which is the target protein. However, unlike a substrate, a substrate inhibitor must not be catalyzed by an enzyme, or it must have a very slow reaction rate even when catalyzed. Such substances include competitive inhibitors having a structure similar to that of the target protein substrate, and pseudo-substrates. These substrate inhibitors have high specificity for binding to the target protein. Therefore, high target recognition characteristics can be imparted to the imprint polymer. In addition, since it is not catalyzed or the reaction rate is very slow, the binding of the target protein to the substrate recognition site is stable.
[0049] なお、基質阻害剤の親和性は特に限定されないが、 103M以上であることが好まし い。  [0049] Although the affinity of the substrate inhibitor is not particularly limited, it is preferably 103M or more.
[0050] 例えばリボヌクレアーゼを標的タンパク質とする場合、基質阻害剤としては、各種ヌ クレオチドが利用できる。また、ヌクレオチドにさらにビニル基を有するものならば、付 加重合が容易に行え、その固定ィ匕量を制御できるため、好ましい。 DNA類似体など もこれに含まれる。図 5に、スチレン修飾リボヌクレアーゼ阻害剤（2， -UMP、および 3， -UMP)の構造式を示す。  [0050] For example, when ribonuclease is used as a target protein, various nucleotides can be used as a substrate inhibitor. In addition, it is preferable that the nucleotide further has a vinyl group because addition polymerization can be easily performed and the amount of fixation can be controlled. This includes DNA analogs. FIG. 5 shows the structural formulas of styrene-modified ribonuclease inhibitors (2, -UMP and 3, -UMP).
[0051] 本発明のインプリントポリマーは、このような基質阻害剤を、単独で備えてもよいし、 上記（ 1 3)欄の金属錯体と共に備えてもよ!、。金属錯体と組み合わせて備えること により、インプリントポリマーは協同的な効果により高い親和性を得ることができる。  [0051] The imprint polymer of the present invention may be provided with such a substrate inhibitor alone or with the metal complex in the above-mentioned (13) column! By providing it in combination with a metal complex, the imprint polymer can obtain high affinity due to a cooperative effect.
[0052] (1 5)コモノマー  [0052] (1 5) Comonomer
本発明に係るインプリントポリマーは、上記（1 1)および（1— 2)に記載した金属 錯体および基質阻害剤の少なくとも一方を機能性モノマーとして含むと共に、これら 機能性モノマーと共重合するコモノマーを含んで 、てもよ 、。機能性モノマーとともに 共重合させるコモノマーには、上記（1 1)で述べた機能性モノマーを利用すること ができる。 MPC([2- (methacryloyloxy)ethyl]phosphorylcholine)ゝ又は GEMA(Glycosylo xyethyl methacrylate)を添加することにより、非特異的相互作用を低減させる。 The imprint polymer according to the present invention contains, as a functional monomer, at least one of the metal complex and the substrate inhibitor described in the above (11) and (1-2). Including comonomers that copolymerize with functional monomers. As the comonomer to be copolymerized with the functional monomer, the functional monomer described in the above (11) can be used. Addition of MPC ([2- (methacryloyloxy) ethyl] phosphorylcholine) or GEMA (Glycosyloxyethyl methacrylate) reduces non-specific interactions.
[0053] < 2.製造方法 >  [0053] <2. Manufacturing method>
本発明に係るインプリントポリマーの製造方法は、標的タンパク質と機能性モノマー との複合体を形成する複合体形成工程と、上記複合体中の機能性モノマーを重合さ せる重合工程とを含む。  The method for producing an imprint polymer according to the present invention includes a complex forming step for forming a complex of a target protein and a functional monomer, and a polymerization step for polymerizing the functional monomer in the complex.
[0054] 本発明に係る製造方法の一例を、図 1に基づいて説明する。  An example of the manufacturing method according to the present invention will be described with reference to FIG.
[0055] まず、図 1の（a) , (b)に示すように、標的タンパク質と、当該標的タンパク質に配位 結合可能な金属および当該金属を配位する配位子力もなる金属錯体とを、上記金属 を介して会合させる（複合体形成工程)。このとき、ポリマーの合成系には、図示しな V、重合可能な機能性モノマーが含まれて 、る。  [0055] First, as shown in (a) and (b) of FIG. 1, a target protein, a metal capable of coordinating and binding to the target protein, and a metal complex that also has a ligand force to coordinate the metal. Then, they are associated via the metal (complex formation step). At this time, the polymer synthesis system includes V, not shown, and a polymerizable functional monomer.
[0056] 次に、図 1の（c)に示すように、機能性モノマーを重合させることによって、金属錯体 を標的タンパク質と相補的な位置に固定する (重合工程)。  Next, as shown in FIG. 1 (c), the functional complex is polymerized to fix the metal complex at a position complementary to the target protein (polymerization step).
[0057] 次に、図 1の（d)に示すように、標的タンパク質を除去する。  Next, as shown in FIG. 1 (d), the target protein is removed.
[0058] このようにして、標的タンパク質を認識する認識部位をもつインプリントポリマーを製 造する。  [0058] In this way, an imprinted polymer having a recognition site that recognizes a target protein is produced.
[0059] (2— 1)複合体形成工程 (図 1の (b) )  [0059] (2-1) Complex formation process ((b) in Fig. 1)
複合体形成工程は、標的タンパク質と機能性ポリマーとを混合すればよぐその他 の条件は、従来のインプリントポリマーの製造方法を好適に利用可能である。  In the complex formation step, a conventional method for producing an imprinted polymer can be suitably used as other conditions in which the target protein and the functional polymer are mixed.
[0060] 機能性モノマーとタンパク基質との複合体形成は水中で行うことが望ま 、。 [0060] The complex formation between the functional monomer and the protein substrate is preferably performed in water.
[0061] 用いることのできる機能性モノマーとしては、上記（1— 1)で説明した通りである。 [0061] Functional monomers that can be used are as described in (1-1) above.
[0062] 複合体形成工程において特に好適な条件としては、タンパク質濃度が数 100 M 、機能性モノマーが 2当量〜 5等量程度が望ましい（リボヌクレアーゼの場合)。タンパ ク質は会合しない濃度領域にしなければならない。また、機能性モノマーは解離定 数よりも濃 、濃度条件でなければならな 、。温度は室温で行う。 [0062] Particularly preferred conditions in the complex formation step are a protein concentration of several hundred M and a functional monomer of about 2 equivalents to 5 equivalents (in the case of ribonuclease). The protein must be in a concentration range where it does not associate. Also, the functional monomer must be thicker than the dissociation constant and the concentration condition. The temperature is room temperature.
[0063] また、機能性モノマーとして、上記（1 3)の配位子、および、上記（1 4)の基質 阻害剤の少なくとも一方を含む機能性モノマーを用いてもよい。この場合、複合体形 成工程および下記重合工程は、上記（1 5)のコモノマーの存在下で行ってもょ 、。 [0063] Further, as the functional monomer, the ligand of (1 3) above and the substrate of (1 4) above A functional monomer containing at least one of the inhibitors may be used. In this case, the complex formation step and the following polymerization step may be performed in the presence of the comonomer (15) above.
[0064] また、複合体形成工程および重合工程は、架橋剤の存在下で行ってもよ!ヽ。架橋 剤には、メチレンビスアクリルアミド等、比較的水溶性のある化合物が利用できる。ま た、金属配位能を持たないものが好ましい。 [0064] Further, the complex formation step and the polymerization step may be performed in the presence of a crosslinking agent! As the crosslinking agent, a relatively water-soluble compound such as methylenebisacrylamide can be used. Further, those having no metal coordination ability are preferred.
[0065] 基質阻害剤を機能性モノマーとして用いる場合、親和性は 103Mほどあればよい。 [0065] When a substrate inhibitor is used as a functional monomer, the affinity should be about 10 3 M.
例えば、リボヌクレアーゼに対して 2'—CMPは比較的良好な阻害剤である。この特 異的結合を阻害させな 、ためには、結合に関与して 、な 、5 '位に重合性官能基を 導入すればよい。  For example, 2'-CMP is a relatively good inhibitor for ribonucleases. In order not to inhibit this specific bond, a polymerizable functional group may be introduced at the 5 ′ position without being involved in the bond.
[0066] このリン酸ィ匕化合物の類似体は機能性モノマーとして非常に有用である。タンパク 質の分子インプリンティングでは通常選択性が非常に低 、が、この基質阻害剤を用 いることにより、強い相互作用と選択性を与えることができる。カルボン酸などのイオン 性のモノマー、ピリジンなどの水素結合性のモノマー、又は金属錯体など、通常のィ ンプリンティングで用いられる相互作用と組み合わせることにより、親和性を向上させ ることがでさる。  [0066] The analog of the phosphoric acid compound is very useful as a functional monomer. In molecular imprinting of proteins, the selectivity is usually very low, but by using this substrate inhibitor, strong interaction and selectivity can be given. Affinity can be improved by combining with ionic monomers such as carboxylic acids, hydrogen bonding monomers such as pyridine, or interactions used in normal imprinting such as metal complexes.
[0067] 他のタンパク質においても、重合工程によって基質阻害剤を固定ィ匕することにより、 目的タンパク質に対する親和性と特異性を向上させることができる。ぺプチダーゼに 対するペプチド阻害剤、レクチンに対する糖類似体、各種キナーゼに対するリン酸化 ペプチドがそれぞれ対応する。  [0067] For other proteins as well, the affinity and specificity for the target protein can be improved by immobilizing the substrate inhibitor in the polymerization step. Peptide inhibitors for peptidases, sugar analogs for lectins, and phosphorylated peptides for various kinases correspond.
[0068] 金属錯体を機能性モノマーとして利用する場合、不要な金属'イオン等の共雑物は 親和性を下げることになり、認識能を低下させる。ただし、タンパク質が不安定で塩強 度、 pH等の条件がある場合には、それに従う。インプリントの効果は機能性モノマー が標的タンパク質に対して 2分子以上会合した場合に発現するので、機能性モノマ 一の量は標的タンパク質に対して 2当量〜 5等量程度が望ましい。基質阻害剤をモノ マーとして用いた場合には、標的タンパク質に対して 1当量以上でよ!、。  [0068] When a metal complex is used as a functional monomer, unnecessary contaminants such as metal 'ions lower the affinity and lower the recognition ability. However, if the protein is unstable and there are conditions such as salt strength and pH, follow them. Since the imprint effect is expressed when two or more functional monomers associate with the target protein, the amount of the functional monomer is preferably about 2 to 5 equivalents with respect to the target protein. If a substrate inhibitor is used as a monomer, it should be at least 1 equivalent to the target protein!
[0069] 複合体形成および重合工程では、用 ヽる緩衝液は金属配位を阻害する緩衝液で なければよい。リン酸緩衝液は亜鉛錯体などに対して阻害能を示すため、好ましくな い場合がある。同様の理由から、 HEPES緩衝液よりもトリス (Tris)緩衝液の方力 金属 配位能が低い場合が多ぐ好ましい。金属配位を阻害しない限りにおいては、炭酸' ホウ酸 ·酢酸 ·リン酸緩衝液が利用可能である。 [0069] In the complex formation and polymerization step, the buffer solution used may not be a buffer solution that inhibits metal coordination. Phosphate buffer solution may be undesirable because it exhibits inhibitory activity against zinc complexes and the like. For the same reason, the Tris buffer solution is more powerful than the HEPES buffer. Many cases where the coordination ability is low are preferred. As long as the metal coordination is not inhibited, a carbonic acid boric acid / acetic acid / phosphate buffer solution can be used.
[0070] (2— 2)重合工程（図 1の（c) )  [0070] (2-2) Polymerization process ((c) in Fig. 1)
重合反応は、重合開始剤（単に開始剤と称する場合もある）を添加することによって 、上記 (2— 1)の複合体形成工程で形成された複合体中の機能性モノマーを重合す る工程である。  The polymerization reaction is a step of polymerizing the functional monomer in the complex formed in the complex formation step of (2-1) above by adding a polymerization initiator (sometimes referred to simply as an initiator). It is.
[0071] 重合方法としては、 UV光による重合と熱重合が両方可能である。開始剤には、ァゾ 化合物系の開始剤、または過硫酸カリウムや過硫酸アンモ-ゥムなどの Redox開始 剤が利用できる。これと重合促進剤である TEMEDをカ卩えることにより、温和な条件で も重合反応が進行する。  [0071] As the polymerization method, both polymerization by UV light and thermal polymerization are possible. As the initiator, an azo compound-based initiator or a Redox initiator such as potassium persulfate or ammonium persulfate can be used. By combining this with TEMED, which is a polymerization accelerator, the polymerization reaction proceeds even under mild conditions.
[0072] なお、上記 (2— 1)の複合体形成工程で形成された複合体を、複合体形成工程を 行った水溶液中力も単離し、その後に重合工程を行うことが好ましい。  [0072] Note that it is preferable that the complex formed in the complex forming step (2-1) is also isolated from the aqueous solution in which the complex forming step is performed, and then the polymerization step is performed.
[0073] 例えば、金属錯体を用いた場合の重合工程は、タンパク質一金属錯体複合体を単 離し、その後に行うことにより、高選択的な認識マテリアルの調製が可能である。複合 体を単離するには、結合の強い金属錯体 ·又は解離速度が遅い金属 (Pdなど）を用 いる必要がある。そのためには、金属錯体の複核化は有効である。単離の際の分離 方法は、ゲルろ過、 HPLC、透析など一般的な分離方法が可能である。その後、この 複合体を凍結乾燥することにより単離することができる。  [0073] For example, the polymerization step in the case of using a metal complex can be carried out after the protein-metal complex complex is isolated, whereby a highly selective recognition material can be prepared. In order to isolate the complex, it is necessary to use a metal complex with a strong bond or a metal with a low dissociation rate (such as Pd). For this purpose, binucleation of metal complexes is effective. As a separation method at the time of isolation, general separation methods such as gel filtration, HPLC, and dialysis can be used. The complex can then be isolated by lyophilization.
[0074] また、標的タンパク質の非特異的な結合を抑制することにより選択性を向上させるこ とがでさる。  [0074] In addition, selectivity can be improved by suppressing non-specific binding of the target protein.
[0075] 具体的には、複合化工程後、または重合工程を行った後に、機能性モノマー中（重 合工程後はインプリントポリマー中）の金属錯体中の金属に、この金属に結合可能な 配位子を結合させる。铸型タンパク質と相補的な位置に正確に配置されて 、な 、金 属錯体があると、非特異的な結合が起こる。上記ブロック剤によると、このような正確 に配置されていない金属錯体をブロックすることができるので、特異性が向上する。こ のようなブロック剤としては、エチレンジァミンの他、 EDTAやイミノジ酢酸、ユトリロ三 酢酸などのキレート剤、各種ポリアミンが利用できる。  [0075] Specifically, after the complexing step or the polymerization step, the metal in the metal complex in the functional monomer (after the polymerization step and in the imprint polymer) can bind to this metal. Bind the ligand. If there is a metal complex that is precisely located at a position complementary to the saddle protein, non-specific binding occurs. According to the blocking agent, such a metal complex that is not accurately arranged can be blocked, so that specificity is improved. As such a blocking agent, in addition to ethylenediamine, chelating agents such as EDTA, iminodiacetic acid and utrilotriacetic acid, and various polyamines can be used.
[0076] (2— 3)铸型タンパク質の除去（図 1の（d) ) 铸型タンパク質の除去は水 ·緩衝液により行う。これらの溶液により数時間程度洗浄 することにより铸型分子は洗い流される。それでも除去されない場合は、 lOOmMの N aClで 5分間洗浄することにより除去される。それでも完全に洗い流されない場合は、 EDTAなどのキレート剤を添加し、一度金属を洗い流し、これにより強制的にタンパ ク質を除去する。この後再度金属を添加し、認識部位を再構築する。 [0076] (2-3) Removal of vertical protein ((d) in Fig. 1) Use a water / buffer solution to remove the saddle protein. By washing with these solutions for several hours, the cage molecules are washed away. If it is still not removed, it can be removed by washing with lOOmM NaCl for 5 minutes. If it still does not wash away completely, add a chelating agent such as EDTA, wash away the metal, and forcibly remove the protein. Thereafter, the metal is added again to reconstruct the recognition site.
[0077] なお上記の製造方法は、バルタ重合の他、様々な表面上に導入できる。ポリスチレ ン榭脂、シリカ粒子、カーボンナノチューブ上、ガラス基板上、金基板上、各種金属 基板上等、重合性官能基を導入できる材料上ならば制限を受けることはない。  [0077] The above production method can be introduced on various surfaces in addition to the Balta polymerization. There are no restrictions on materials that can introduce polymerizable functional groups, such as polystyrene resin, silica particles, carbon nanotubes, glass substrates, gold substrates, and various metal substrates.
[0078] < 3.アレイチップ >  [0078] <3. Array chip>
(3— 1)アレイチップの構成  (3-1) Array chip configuration
本発明に係るアレイチップは、上記 < 1 >欄のインプリントポリマーを異なるタンパク 質を铸型にして製造し、多数備えればよぐ他の構成、基板等は特に限定されない。 また、基板上への固定方法も特に限定されるものではなぐ従来の薄膜形成技術や プロテインアレイにおける技術を適用することができる。  The array chip according to the present invention is not particularly limited to other structures, substrates, etc., as long as the imprinted polymer in the <1> column is produced using different proteins in a bowl shape and provided in large numbers. In addition, the fixing method on the substrate is not particularly limited, and conventional thin film forming technology and protein array technology can be applied.
[0079] アレイチップでは、インプリントポリマーは、色素修飾モノマーを有することが好まし い。色素修飾モノマーを、クマリン 'ダンシル 'FITC 'アントラセンなどを蛍光色素とす ることにより、容易にタンパク質を検出することができる。検出方法としては、蛍光スぺ タトル変化 ·蛍光寿命測定 ·蛍光エネルギー移動が可能である。色素は、単に重合性 官能基を導入したモノマーを共重合させる方法、重合した高分子に吸着させる方法 、金属錯体と共有結合させて認識部位の近傍に導入する方法の 3通りがある。  [0079] In the array chip, the imprint polymer preferably has a dye-modifying monomer. Proteins can be easily detected by using dye-modifying monomers such as coumarin 'dansyl' FITC 'anthracene as fluorescent dyes. Possible detection methods include fluorescence spectrum change, fluorescence lifetime measurement, and fluorescence energy transfer. There are three types of dyes: a method in which a monomer having a polymerizable functional group introduced is copolymerized, a method in which it is adsorbed on a polymerized polymer, and a method in which it is covalently bonded to a metal complex and introduced near the recognition site.
[0080] <4.インプリントポリマーを利用したタンパク質の同定方法 >  [0080] <4. Protein identification method using imprint polymer>
本発明に係るタンパク質の同定方法および精製方法は、上記く 1 >欄のインプリン トポリマーへの吸着特性の違 、を利用したものであればょ 、。この際に異なった錯体 、铸型タンパク質、金属、架橋比で調製したインプリントポリマーのアレイを作製するこ とにより、ノターン解析を行うことができる。  The protein identification method and purification method according to the present invention should utilize the difference in the adsorption property to the imprint polymer in the column 1> above. In this case, noturn analysis can be performed by preparing an array of imprinted polymers prepared with different complexes, cage proteins, metals, and crosslinking ratios.
[0081] 具体的には、上記 < 3 >欄のアレイチップを利用し、その色素修飾モノマーの発す るシグナルを検出、解析することで、タンパク質を同定することができる。さらに、デコ ンポリューションにより、細胞や組織力も複数のタンパク質を定量的に検出することも 可能となる。 [0081] Specifically, the protein can be identified by using the array chip in the <3> column and detecting and analyzing the signal emitted by the dye-modified monomer. In addition, deconvolution can also detect multiple proteins quantitatively in cells and tissue forces. It becomes possible.
[0082] < 5.リフォールデイング方法 >  [0082] <5. Refolding method>
タンパク質の認識をリフォールデイングに利用することも可能である。すなわち、特 定のタンパク質のうち、ある 3次構造の状態をとつているタンパク質だけを認識するこ とが可能である。タンパク質は変性状態の時は通常のびた状態をとつており、天然状 態では折りたたまれている。この天然状態をインプリントすることにより、天然状態のタ ンパク質のみを結合することができ、タンパク質の折りたたみを促進することとなる。  Protein recognition can also be used for refolding. In other words, it is possible to recognize only proteins that have a certain tertiary structure state among specific proteins. Proteins are normally stretched when in a denatured state and are folded in their natural state. By imprinting this natural state, only the protein in the natural state can be bound and protein folding is promoted.
[0083] リフォールデイング方法としては、具体的には、再生緩衝液中で、変性タンパク質と インプリントポリマーとを接触させればよ!、。  [0083] As a refolding method, specifically, a denatured protein and an imprinted polymer may be brought into contact with each other in a regeneration buffer!
[0084] (再生緩衝液）  [0084] (Regeneration buffer)
再生緩衝液とは、上記で調製したインプリントポリマーを含む緩衝溶液である。例え ば、 10mg/mLのインプリントポリマー、および 50mMの NaClを含み pH7. 4のトリ ス緩衝液等を用いることができる。  The regeneration buffer is a buffer solution containing the imprint polymer prepared above. For example, a 10 mg / mL imprint polymer and a Tris buffer solution containing 50 mM NaCl and pH 7.4 can be used.
[0085] (接触方法）  [0085] (Contact method)
変性タンパク質とインプリントポリマーとを接触させる方法としては、インプリントポリ マーを固定しない状態で用いるバッチ法、および、インプリントポリマーをカラム等の 形状に固定して用いる flow法がある。接触方法としては、標的タンパク質やその他条 件に合わせて適宜設定可能である。ノ ツチ法では、タンパク質一ポリマー複合体とな るので、数時間のリフォールデイングの後、塩や pHなどにより解離させればターゲット タンパク質を回収できる。 Flow法においては、天然状態にフォールデイングしたタン パク質のみが強く結合するため、折りたたまれずに早く溶出してきたものは再度、変 性ーリフォールデイングを行えばょ 、。  As a method of bringing the denatured protein into contact with the imprint polymer, there are a batch method in which the imprint polymer is not fixed, and a flow method in which the imprint polymer is fixed in a shape such as a column. The contact method can be appropriately set according to the target protein and other conditions. The Notch method results in a protein-polymer complex, so that after several hours of refolding, the target protein can be recovered by dissociation with salt or pH. In the Flow method, only the protein that has been folded into the natural state binds strongly, so if it elutes quickly without being folded, re-fold it again.
[0086] 天然状態のインプリンティングだけでなぐタンパク質の融解状態 (モルテン'グロビ ユール状態)をインプリントすることにより、得られた高分子がタンパク質の凝集を阻害 し、タンパク質の折りたたみを促進することもできる。  [0086] By imprinting the melted state of the protein (Morten's globule state) just by imprinting in the natural state, the resulting polymer can inhibit protein aggregation and promote protein folding. it can.
[0087] 変性状態タンパク質、すなわち非天然状態タンパク質の調製は、外部からの熱や 変性剤添カ卩により行う。リボヌクレアーゼならば 80°C〜90°Cで完全に変性する。これ に上記金属錯体を添加し、熱重合により変性タンパク質特異的なインプリントポリマ 一が可能となる。得られたポリマーは、変性状態の検出やタンパク質除去へと利用で きる。変性剤により変性させたタンパク質のインプリンティングも同様に可能である。 [0087] A denatured state protein, that is, a non-natural state protein is prepared by heat from the outside or a denaturant-added agent. Ribonuclease is completely denatured at 80 ° C to 90 ° C. The above-mentioned metal complex is added to this, and the imprinted polymer specific to the denatured protein is obtained by thermal polymerization. One is possible. The obtained polymer can be used for detection of denatured state and protein removal. Imprinting of proteins denatured with a denaturant is also possible.
[0088] また、上記方法を利用して、タンパク質のおりたたみ状態を、結合親和性の差から 蛍光色素で検出するアレイを構築することができる。  [0088] Further, by using the above method, it is possible to construct an array that detects the state of protein collapse with a fluorescent dye from the difference in binding affinity.
[0089] < 6.タンパク質固定化法 >  [0089] <6.Protein immobilization method>
タンパク質の認識能を落とさずに非共有結合的に固定ィ匕する方法として、本発明 のインプリントポリマーの製造方法が利用できる。  The imprint polymer production method of the present invention can be used as a method for non-covalent immobilization without degrading protein recognition ability.
[0090] そのためにまず、標的タンパク質を、当該タンパク質に対して特異的に結合する基 質とまず複合ィ匕させる。リボヌクレアーゼの場合、 PH5. 5で 2' -CMPと複合化させる ことにより、 1： 1で完全に複合化する。さらにこれを上記 < 2>と同様に、インプリント することで、基質認識部位をブロックした状態でタンパク質を固定ィ匕することができる [0090] For this purpose, first, a target protein is first complexed with a substrate that specifically binds to the protein. For ribonuclease, by P H5 5 with 2 '-CMP complexed with, 1:. Completely complexed with 1. Furthermore, by imprinting this as in <2> above, the protein can be immobilized with the substrate recognition site blocked.
[0091] この場合、競争的に金属がタンパク質一基質相互作用を阻害しないように、 K〜10 3Μ程度の親和性の結合を有する金属錯体を用いる。固定化した後、中性〜弱塩基 性、及び高塩濃度条件にすることにより特異的なリガンド（2'— CMP)を解離させるこ とができる。得られた高分子は、機能性モノマーが基質近傍に結合せずに固定化さ れるため、天然状態の基質認識能を保持したしたままセンシング、又は分離材料とし て利用できる。 [0091] In this case, a metal complex having an affinity of about K to 10 3 Μ is used so that the metal does not competitively inhibit the protein-substrate interaction. After immobilization, the specific ligand (2'-CMP) can be dissociated by using neutral to weakly basic and high salt conditions. The obtained polymer is immobilized without binding the functional monomer in the vicinity of the substrate, so that it can be used as a sensing or separation material while retaining the substrate recognition ability in the natural state.
[0092] 以上のように、本発明に係るインプリントポリマーは、標的タンパク質が可逆的に結 合する認識部位を持ち、上記認識部位は、金属錯体および Ζまたは基質阻害剤を 有する。これにより、従来のインプリントポリマーと比較して特異性が向上しており、よ り正確にタンパク質を同定することができ、センサーアレイへの応用により、さらに正 確な同定と混合物の組成の解析を可能として 、る。  [0092] As described above, the imprint polymer according to the present invention has a recognition site to which a target protein binds reversibly, and the recognition site has a metal complex and a cage or a substrate inhibitor. As a result, specificity is improved compared to conventional imprinted polymers, proteins can be identified more accurately, and application to sensor arrays enables more accurate identification and analysis of the composition of the mixture. Possible.
[0093] また、本発明に係るインプリントポリマーの製造方法は、標的タンパク質と結合可能 な金属を配位する金属錯体、および Ζまたは、標的タンパク質の基質阻害剤を機能 性モノマーとして用い、上記機能性モノマーを固定ィ匕する固定工程を含むことを特徴 としている。  [0093] Further, the method for producing an imprint polymer according to the present invention uses a metal complex that coordinates a metal capable of binding to a target protein, and a substrate or a substrate inhibitor of the target protein as a functional monomer. It is characterized by including a fixing step for fixing the functional monomer.
[0094] 金属配位による特異的結合は、非特異的な結合、すなわちタンパク質の結合部位 をうまく形成できな力つた金属錯体の配位結合能を消滅させることにより、顕在化させ ることが可能である。そのために、タンパク質と金属錯体を複合化させた後、又はこれ を固定化させた後にキレート剤等を添加することにより、非特異的結合に関与する余 分な金属をマスキングし、非特異的な配位結合を低下させることができる。このような 正確な特異性の発現は水素結合、イオン結合と!/、つた従来のインプリンティング法に はなかったものであり、金属配位特有のものである。 [0094] Specific binding by metal coordination is non-specific binding, that is, a protein binding site. It can be made clear by eliminating the coordination ability of a powerful metal complex that cannot be formed successfully. Therefore, after complexing the protein with the metal complex or immobilizing it, the addition of a chelating agent or the like masks the extra metal involved in non-specific binding, and the non-specific binding. Coordination bonds can be reduced. Such precise expression is unique to metal coordination, which is not possible with conventional imprinting methods such as hydrogen bonding, ionic bonding, and! /.
[0095] 種々のタンパク質インプリントポリマーを合成してインプリントポリマーアレイとし、そ れに対して複数のタンパク質を再結合させ、各タンパク質の各ポリマーに対する結合 量のパターンを主成分分析して 2, 3次元のデータに変換したところ、タンパク質混合 液は完全に成分の組成の分類分けがなされた。これは、複雑な試料に対して、イン プリントポリマーアレイを用いることで、含まれるタンパク質の同定が可能なことを示し ている。また、未知のタンパク質のクラス分けも可能で、有用な機能をもつタンパク質 のスクリ一ユングや、タンパク質組成のプロフアイリングに威力を発揮する。  [0095] Various protein imprinted polymers were synthesized to form an imprinted polymer array, a plurality of proteins were recombined with each other, and the amount of binding of each protein to each polymer was analyzed by principal component analysis. When converted to three-dimensional data, the protein mixture was completely classified into component compositions. This indicates that the protein contained in the imprinted polymer array can be identified for complex samples. It is also possible to classify unknown proteins, which is very useful for screening proteins with useful functions and profiling protein compositions.
[0096] タンパク質は、折りたたみ構造をとることで機能を発現する。そこで、本発明のイン プリントポリマーの分子認識能を用いると、うまく折りたたまれな力つたタンパク質を折 りたたみ構造との複合ィ匕により安定ィ匕し、正しい折りたたみ構造に変換できる。人工 的にタンパク質を生産するときに、生産したポリペプチドを正しく折りたたみ、機能を 発現させることはバイオテクノロジーの分野では特に重要であり、本発明は、タンパク 質の同定や分離の手法を提供するのみならず、タンパク質の折りたたみ構造を誘導 する人工分子シャペロンの役割も果たす。  [0096] A protein expresses a function by taking a folded structure. Therefore, by using the molecular recognition ability of the imprinted polymer of the present invention, a protein that has been successfully folded can be stabilized by a complex structure with the folded structure and converted into a correct folded structure. When a protein is artificially produced, it is particularly important in the biotechnology field to correctly fold the produced polypeptide and express its function, and the present invention only provides a method for protein identification and separation. In addition, it plays the role of an artificial molecular chaperone that induces protein folding.
[0097] また、タンパク質は条件により、 2次構造として a 一へリックスや /3 シートをとり、こ れらは全体の高次構造に大きく影響を与え、時には疾病の原因ともなる。近年、 BSE 問題など、これらタンパク質の高次構造の検出'判別が大きな課題となっている。こう した問題についても、本発明のタンパク質インプリンティングは解決法を提供し、ぺプ チドの折りたたみ状態を判別する素子を製造することもできる。上記構成により、抗体 等の天然の化合物よりも簡便な方法によって、標的タンパク質を認識可能な物質を 製造することができる。  [0097] Depending on the conditions, proteins take a one helix or / 3 sheet as a secondary structure, which greatly affects the overall higher order structure and sometimes causes disease. In recent years, the detection and discrimination of higher-order structures of these proteins, such as the BSE problem, has become a major issue. With respect to these problems, the protein imprinting of the present invention provides a solution, and it is also possible to produce an element for discriminating the folded state of the peptide. With the above configuration, a substance capable of recognizing the target protein can be produced by a simpler method than natural compounds such as antibodies.
[0098] なお、本発明は上述した実施形態に限定されるものではなぐ請求項に示した範囲 で種々の変更が可能である。すなわち、請求項に示した範囲で適宜変更した技術的 手段を組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる [0098] It should be noted that the present invention is not limited to the above-described embodiments, but is within the scope indicated in the claims. Various changes can be made. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.
[0099] 以下、実施例により、本発明をさらに詳細に説明する力 本発明はこれらにより何ら 限定されるものではない。 [0099] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
[0100] (実施例） [0100] (Example)
〔I〕金属錯体を有するインプリントポリマーとその利用  [I] Imprint polymer having metal complex and its use
[1]サイクレン修飾スチレンの合成（図 6)  [1] Synthesis of cyclene-modified styrene (Figure 6)
金属錯体機能性モノマーの合成を以下の工程で行った。  The metal complex functional monomer was synthesized by the following steps.
[0101] [1 - 1] (Boc) -cyclenの合成 [0101] Synthesis of [1-1] (Boc) -cyclen
3  Three
サイクレン 4塩酸塩 500 mgを塩化メチレンに溶解し、トリェチルァミン 5当量存在 下、 0°Cに冷却した後、塩化メチレンに溶解した (Boc) 0 3.0当量をゆっくり添加した  Cyclene tetrahydrochloride 500 mg was dissolved in methylene chloride, cooled to 0 ° C in the presence of 5 equivalents of triethylamine, and then 03.0 equivalents of (Boc) dissolved in methylene chloride was slowly added.
2  2
。一昼夜室温で攪拌した後、飽和食塩水で洗浄した。硫酸ナトリウムで有機相を洗浄 した後、シリカゲルクロマトグラフィー（Hexane :AcOEt = 2:1→ 1:2)で精製した。 - NMRにより同定を行った。  . The mixture was stirred overnight at room temperature, and then washed with saturated brine. The organic phase was washed with sodium sulfate and purified by silica gel chromatography (Hexane: AcOEt = 2: 1 → 1: 2). -Identification by NMR.
[0102] [1 - 2] Boc3- cyclen- Stの合成 [0102] [1-2] Synthesis of Boc3- cyclen- St
上記 [1— 1]で得られた Boc3-cyclen 260mgをァセトニトリル中において、 4-vinylben zyl chloride 1. 5当量、炭酸カリウム 2当量を添カ卩し、ヨウ化カリウム 1当量存在下、 65 °Cで 3時間攪拌した。 TLCにて原料の消失を確認し、反応終了後そのまま飽和食塩 水で 3回洗浄した。硫酸ナトリウムで乾燥、溶媒留去の後、シリカゲルクロマトグラフィ 一（Hexane :AcOEt = 3： 2→ 1： 1)により精製を行った。 320 mgゝ 96 %。 1H- NMR(C DC1 ): δ 7.49 (d, 2H, J = 9.4 Hz, ArH), 7.36 (d, 2H, J = 9.4 Hz, ArH), 6.67 (dd, 1 Add 260 mg of Boc3-cyclen obtained in [1-1] above in acetonitrile, add 1.5 equivalents of 4-vinylbenzyl chloride and 2 equivalents of potassium carbonate, and in the presence of 1 equivalent of potassium iodide, 65 ° C For 3 hours. The disappearance of the raw material was confirmed by TLC, and after completion of the reaction, the raw material was washed three times with saturated saline. After drying with sodium sulfate and evaporation of the solvent, purification was performed by silica gel chromatography (Hexane: AcOEt = 3: 2 → 1: 1). 320 mg ゝ 96%. 1H-NMR (C DC1): δ 7.49 (d, 2H, J = 9.4 Hz, ArH), 7.36 (d, 2H, J = 9.4 Hz, ArH), 6.67 (dd, 1
3 Three
H, J = 12.2, 19.5 Hz, ArCH=CH ), 5.9 (br, 1H, NHCOO), 5.68 (d, 1H, J = 19.5 Hz  H, J = 12.2, 19.5 Hz, ArCH = CH), 5.9 (br, 1H, NHCOO), 5.68 (d, 1H, J = 19.5 Hz
2  2
, ArCH=CH ), , 5.2 (s, 1H, NHCOO), 5.19 (d, 1H, J = 12.2 Hz, ArCH=CH ), 4.30 (  , ArCH = CH),, 5.2 (s, 1H, NHCOO), 5.19 (d, 1H, J = 12.2 Hz, ArCH = CH), 4.30 (
2 2 br, 1H, NHCH(CO)CH2), 3.6 (m, 2H, CHCH NH), 1.48 (s, 9H, C(CH ) ), 1.45 (s, 9  2 2 br, 1H, NHCH (CO) CH2), 3.6 (m, 2H, CHCH NH), 1.48 (s, 9H, C (CH)), 1.45 (s, 9
2 3 3  2 3 3
H, C(CH ) ).  H, C (CH)).
3 3  3 3
[1 - 3] Cyclen- Stの合成  [1-3] Synthesis of Cyclen-St
上記 [1 2]で得られた Boc3-Cyclen-St 300 mgを塩化メチレン 10 mLに溶解した 。これに TFA 2 mLを添カ卩し、室温で 3時間撹拌した。 TLCで反応終了を確認し、溶 媒を減圧留去後、エタノール/ジェチルエーテルで再結晶を行った。一次晶 120 mg を減圧乾燥し、目的物 Cyclen- St'2TFA塩を得た。1!" I- NMR(CD OD): δ 2.8- 3.1(m, 300 mg of Boc3-Cyclen-St obtained in [1 2] above was dissolved in 10 mL of methylene chloride. . To this was added 2 mL of TFA, and the mixture was stirred at room temperature for 3 hours. After confirming the completion of the reaction by TLC, the solvent was distilled off under reduced pressure, and then recrystallization was performed with ethanol / jetyl ether. 120 mg of primary crystals were dried under reduced pressure to obtain the target product, Cyclen-St'2TFA salt. 1 ! "I-NMR (CD OD): δ 2.8- 3.1 (m,
3  Three
8H, CH of cyclen), d 3.2—3.4 (m, 8H, CH of cyclen), 3.83 (s, 2H, AeCH ), 5.25 (d, 8H, CH of cyclen), d 3.2—3.4 (m, 8H, CH of cyclen), 3.83 (s, 2H, AeCH), 5.25 (d,
2 2 2 2 2 2
1H, J = 12.2 Hz, ArCH=CH ), 5.79 (d, 1H, J = 19.5 Hz, ArCH=CH ), 6.73 (dd, 1H,  1H, J = 12.2 Hz, ArCH = CH), 5.79 (d, 1H, J = 19.5 Hz, ArCH = CH), 6.73 (dd, 1H,
2 2  twenty two
J = 12.2, 19.5 Hz, ArCH=CH ), 7.33 (d, 2H, J = 8.9 Hz, ArH), 7.46 (d, 1H, J = 8.8  J = 12.2, 19.5 Hz, ArCH = CH), 7.33 (d, 2H, J = 8.9 Hz, ArH), 7.46 (d, 1H, J = 8.8
2  2
Hz, ArH)  Hz, ArH)
[l -4]Cu[cyclen-St]Clの合成  Synthesis of [l-4] Cu [cyclen-St] Cl
2  2
上記 [1 3]で得られた 1次晶 Cyclen-St'2TFA 63.3 mgを水 20mLに加えた。さらに これに NaOHをカ卩え、塩化メチレンで抽出 (x2)することにより、 TFAの脱塩を行った。有 機相を硫酸ナトリウムで脱水した後、溶媒減圧留去、乾燥を行った。得られた油状ィ匕 合物をエタノール 5 mLに溶解し、これに 2mL (水/ EtOH = 1/1)に溶解した 1当量の 塩化銅 (Π)を滴下した。 60°Cで 1時間撹拌した後、エタノールを減圧留去した。さらに 水 5 mLを添加し、凍結乾燥し、深紫色の結晶を得た (収量 35.2 mg)。同様の方法 により、 Mn · Ni · Co · Zn等の遷移金属にお!/、ても同様にして錯体を得ることができた。  63.3 mg of the primary crystal Cyclen-St'2TFA obtained in [1 3] above was added to 20 mL of water. Further, NaOH was added to this, and TFA was desalted by extraction (x2) with methylene chloride. The organic phase was dehydrated with sodium sulfate, and then the solvent was distilled off under reduced pressure and dried. The obtained oily compound was dissolved in 5 mL of ethanol, and 1 equivalent of copper chloride (Π) dissolved in 2 mL (water / EtOH = 1/1) was added dropwise thereto. After stirring at 60 ° C for 1 hour, ethanol was distilled off under reduced pressure. Further, 5 mL of water was added and freeze-dried to obtain deep purple crystals (yield 35.2 mg). By the same method, a complex could be obtained in the same way for transition metals such as Mn · Ni · Co · Zn!
[2]エチレンジァミン修飾スチレンの合成（図 7)  [2] Synthesis of ethylenediamine-modified styrene (Figure 7)
[2— 1]TFA- DAP(TFA)OHの合成  [2-1] Synthesis of TFA-DAP (TFA) OH
DAP塩酸塩 0.8 gをは力りとり、 MeOH 10 mLを添カ卩した。これにトリエチノレアミンを 5 等量添加し、さらに TFA-OEtを 5当量添加して一昼夜撹拌した。数時間は白濁した ままであつたが、その後透明となった。反応終了後、溶媒留去した後に酢酸ェチルに 溶解し、飽和食塩水'クェン酸水溶液で洗浄した。硫酸ナトリウムで乾燥した後に溶 媒留去し、目的の白色固体 (フレーク状ィ匕合物) TFA-DAP(TFA)- OHを得た。 1.7g, 収率 100 %。 1H-NMR(CD OD): δ 4.68— 4.73(m, 1H, - CHCH -), 3.59—3.74, (m, 2H,  DAP hydrochloride 0.8 g was taken off and MeOH 10 mL was added. To this was added 5 equivalents of triethinoleamine, and 5 equivalents of TFA-OEt was added and stirred overnight. It remained cloudy for several hours, but then became transparent. After completion of the reaction, the solvent was distilled off, and the residue was dissolved in ethyl acetate and washed with saturated brine and aqueous citrate solution. After drying with sodium sulfate, the solvent was distilled off to obtain the desired white solid (flaky compound) TFA-DAP (TFA) -OH. 1.7 g, 100% yield. 1H-NMR (CD OD): δ 4.68— 4.73 (m, 1H, -CHCH-), 3.59—3.74, (m, 2H,
3 2  3 2
CHCH ).  CHCH).
2  2
[2- 2] TFA- DAP(TFA)- Stの合成  [2-2] Synthesis of TFA-DAP (TFA) -St
上記 [2— 1]で合成した TFA-DAP(TFA)- OH 150 mg、クロロメチルスチレン 1.2当 量、ヒドロキシスクシイミド 1.5当量を塩化メチレン 10 mLに溶解し、これに WSC 1.5当 量を加えた。さらにこれに DIEA 3当量を添加し、 10時間撹拌した。反応終了後、 Et ^a> ^) [solo] ^呦^ ^エ Κε α )Η顺- HT Θ？ 150 mg of TFA-DAP (TFA) -OH synthesized in [2-1] above, 1.2 equivalents of chloromethylstyrene, 1.5 equivalents of hydroxysuccinimide are dissolved in 10 mL of methylene chloride, and 1.5 equivalents of WSC is added thereto. It was. Further, 3 equivalents of DIEA was added thereto and stirred for 10 hours. After the reaction is complete, Et ^ a> ^) [solo] ^ 呦 ^ ^ d ε ε α) Η 顺-H T Θ?
¾¾簟
Figure imgf000021_0001
¾¾ 簟
Figure imgf000021_0001
fi °-m^ ^ w 粼 ¾¾簟 ^ ^m ^m^ ^ fi ° -m ^ ^ w ¾ ¾¾ 簟 ^ ^ m ^ m ^ ^
Figure imgf000021_0002
に^。 つ止縱、縱 s¾氺 - 士べ 读 ^)Q?^¾$ r ¾¾iH^^。 ί^ ΐ ^ ^^ 虽 ·η ^
Figure imgf000021_0002
To ^.縱 s s¾ 氺-べ 读 ^) Q? ^ ¾ $ r ¾¾iH ^^. ί ^ ΐ ^ ^^ 虽
iMI.e ^-M^n ^ 4 ^ -^W ^ ^目 2 * 、 （ Ό HD / Horn  iMI.e ^ -M ^ n ^ 4 ^-^ W ^ ^ eyes 2 *, (Ό HD / Horn
% S)CH丄。 ^翻 S、 ¾累、止^蹈 峯暴 08邈べ^ / べエ / d
Figure imgf000021_0003
n <s ΐ'ζ。 · ェつ; " [呦^： ] ψ园)ベ % S) CH 丄. ^ Translation S 、 ¾Progress 、 Stop ^ 蹈
Figure imgf000021_0003
n <s ΐ'ζ. · ;; "[呦 ^:] ψ 园) Be
(8园） dnn-(z ¾χ-{ε[χ-ε](8 AXES) dnn- (z ¾χ- {ε [ χ-ε]
^ mwi^ [WHO] Ο^ ^^ ^ ^ ェ、 奮 き uZ · 0つ ·^ Mwi ^ [WHO] Ο ^ ^^ ^ ^ E, Isamu-out u Z · 0 one -
[s- dva ) 酹 )ί¾目、dTコ W 粼
Figure imgf000021_0004
、つ止縱 晷(s- dva) 酹) ί¾, dT KO W 粼
Figure imgf000021_0004
縱
^ DnD^^-、つ ¾缀コ πω e氺 οε ¾-dva^¾ [ε - s] 3ΐτ^ D n D ^^-, ¾ 缀 コ π ω e 氺 οε ¾-dva ^ ¾ [ε-s] 3ΐτ
^Ο) DpS-dVa]nD[^-Z] ^ Ο) DpS-dVa] nD [^-Z]
HDHD 'Ηΐ 'ω) 00·ε '(¾つ Ηつ 'Ηΐ qZ'Z HDHD 'Ηΐ ' ω)99·ε HD^V 'ΗΖ) Vf '( Ηつ： 'Ηΐ ΖΖ' HD=HD^V 'Ηΐ 8Z"S '(HD-i V 'Ηΐ 'ω) 8"9-Z"9 '(HA 'Hf 'ω) WL-ZZ'L 9 ： (αθ^つ) WN- HT °% 86 ¾ί §ω Z s氺 べ
Figure imgf000021_0005
οοΐ— [z - s] 3IT HDHD 'Ηΐ' ω) 00 · ε '(¾ つ Η つ' Ηΐ qZ'Z HDHD 'Ηΐ' ω) 99 · ε HD ^ V 'ΗΖ) Vf' (Η つ: 'Ηΐ ΖΖ' HD = HD ^ V 'Ηΐ 8Z "S' (HD-i V 'Ηΐ' ω) 8" 9-Z "9 '(HA' Hf 'ω) WL-ZZ'L 9: (αθ ^) WN- H T °% 86 ¾ί §ω Z s
Figure imgf000021_0005
οοΐ— [z-s] 3IT
¾^^ s- dva[s— ¾ ^^ s- dva [s—
HDHD ΉΖ 'ΖΗ 8·9 'Ρ) ΐΖ·ε HD^V 'HZ '^)0V HDHD HDHD ΉΖ ' Ζ Η 8 · 9' Ρ) ΐΖ · ε HD ^ V 'HZ' ^) 0V HDHD
'HI 'ΖΗ8·9 ZL'f HD=HD^V 'Ηΐ '¾) ZZ' HD=HD^V 'Ηΐ '¾) 9Z"S '(H V Ή ΐ U'9 '( 'Hf 0VL-6VL 9 :( <3つ) Η顺- Ητ
Figure imgf000021_0006
' OST'HI' ΖΗ8 · 9 ZL'f HD = HD ^ V 'Ηΐ' ¾) ZZ 'HD = HD ^ V' Ηΐ '¾) 9Z "S' (HV Ή ΐ U'9 '(' Hf 0VL-6VL 9 :( <3) Η 顺-Η τ
Figure imgf000021_0006
'OST
Figure imgf000021_0007
(ΐ : S'l ΐ : S = raO。V:3UBxsH)— ^ 4ム 、 H つ粼¾ マ (H 邈^
Figure imgf000021_0008
ひ 99I£/900Zdf/ェ:) d 03 ST6CZ0/.00Z OAV 化合物 2(500mg, 1.7 mmol)に 4- DMAP 41 mg、塩化メチレン 30 mlをカ卩え、氷冷下 、メタンスルフォ-ルクロリド（450 mg, 3.9 mmol)をゆっくり滴下した。室温で 4時間攪 拌し、 TLCで確認したところ、 目的物と思われるスポットが確認されたので、ここで反 応を終了させた。食塩水/塩化メチレンで抽出 (2回)し、硫酸マグネシウムで乾燥し、 溶媒を留去した後、シリカゲルクロマトグラフィー（7 % MeOH/CH C1 )により精製した
Figure imgf000021_0007
(ΐ: S'l ΐ: S = raO. V: 3 UBxs H ) — ^ 4 um, H tsu 粼 ¾ ma (H 邈 ^
Figure imgf000021_0008
99I £ / 900Zdf / e :) d 03 ST6CZ0 / .00Z OAV To Compound 2 (500 mg, 1.7 mmol), 41 mg of 4-DMAP and 30 ml of methylene chloride were added, and methanesulfonyl chloride (450 mg, 3.9 mmol) was slowly added dropwise under ice cooling. The mixture was stirred at room temperature for 4 hours and confirmed by TLC. As a result, a spot that seemed to be the target product was confirmed. The reaction was terminated here. Extraction with brine / methylene chloride (twice), drying over magnesium sulfate, evaporation of the solvent, and purification by silica gel chromatography (7% MeOH / CH C1)
2 2  twenty two
。溶媒を留去した後、真空乾燥させると白色粉末を得た。 1H-NMR(CDC13)にて化合 物 3の生成を確認した。生成量 400mg、収率 64.7%であった。  . After the solvent was distilled off, vacuum drying was performed to obtain a white powder. Formation of Compound 3 was confirmed by 1H-NMR (CDC13). The amount produced was 400 mg, and the yield was 64.7%.
[0106] (化合物 4の合成） [0106] (Synthesis of Compound 4)
化合物 3(400 mg, 1.1 mmol)を乾燥 DMF 15 ml中で、アジ化ナトリウム 230 mg, 3.5 mmolを加え、 45°Cで 22時間攪拌しながら反応させた。反応終了後、溶媒を減圧留 去した後、塩化メチレンに溶解させて、飽和食塩水により洗浄した。硫酸マグネシゥ ムで乾燥を行った後、ろ過した。溶媒を留去し、真空乾燥させた後、シリカゲルクロマ トグラフィー (2 % MeOH I CH C1 )で精製した。溶媒を留去した後、真空乾燥させると  Compound 3 (400 mg, 1.1 mmol) was added with sodium azide 230 mg, 3.5 mmol in 15 ml of dry DMF, and reacted at 45 ° C. with stirring for 22 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved in methylene chloride and washed with saturated brine. After drying with magnesium sulfate, it was filtered. After the solvent was distilled off and vacuum-dried, the residue was purified by silica gel chromatography (2% MeOH I CH C1). After removing the solvent and vacuum drying
2 2  twenty two
無色油状物質を得た。 ^-NMRCCDCl )にて化合物 4の生成を確認した。生成量 300  A colorless oil was obtained. The formation of Compound 4 was confirmed by ^ -NMRCCDCl). Production amount 300
3  Three
mg,収率 87.3 %であった。  mg, yield 87.3%.
[0107] (化合物 5の合成） [0107] (Synthesis of Compound 5)
化合物 4(300 mg, 0.96 mmol)をメタノール約 20 mlに溶解し、パラジウム炭素 150 mg を加え、攪拌しながら水素を投入した。その後、攪拌しながら 16時間反応させ、 TLC( 1 % MeOH I CH CI +アンモニア水 1滴)にて反応の終了を確認した。セライトを補  Compound 4 (300 mg, 0.96 mmol) was dissolved in about 20 ml of methanol, 150 mg of palladium carbon was added, and hydrogen was added while stirring. Thereafter, the reaction was allowed to proceed for 16 hours with stirring, and the completion of the reaction was confirmed by TLC (1% MeOH I CH CI + 1 drop of aqueous ammonia). Supplement with Celite
2 2  twenty two
助剤としてろ過し、溶媒を減圧留去して真空乾燥を行うと無色油状物質を得た。 Filtration as an auxiliary agent, the solvent was distilled off under reduced pressure, and vacuum drying was performed to obtain a colorless oily substance.
NMR(CDC1 )にて化合物 5の生成を確認した。生成量 220 mg, 収率 80.2 %であった Formation of compound 5 was confirmed by NMR (CDC1). Product yield 220 mg, yield 80.2%
3  Three
[0108] (化合物 6の合成） [0108] (Synthesis of Compound 6)
化合物 5を塩化メチレン中、スチレンカルボン酸 1当量、 WSC 1当量、 HOBt 1 当量、 DIEA 1当量を添加し、縮合反応を行った。クェン酸、重曹で洗浄した後、シリ 力ゲルクロマトグラフィーにて精製を行った。  Compound 5 was subjected to a condensation reaction by adding 1 equivalent of styrene carboxylic acid, 1 equivalent of WSC, 1 equivalent of HOBt, and 1 equivalent of DIEA in methylene chloride. After washing with citrate and sodium bicarbonate, purification was performed by silica gel chromatography.
[0109] (化合物 7の合成）  [0109] (Synthesis of Compound 7)
化合物 6を 10 %TFA塩化メチレン溶液にて脱保護し、化合物 7を得た。 [0110] (化合物 8の合成） Compound 6 was deprotected with 10% TFA methylene chloride solution to give compound 7. [0110] (Synthesis of Compound 8)
ホスホロアミダイド試薬を 1当量、テトラゾール存在下、水酸基と反応させた。酸化剤 添加を添加した後、 HPLCで精製を行った。 2'位と 3 '位がリン酸ィ匕されたスチレン修 飾 UMP (化合物 8)を得た。  One equivalent of phosphoramidide reagent was reacted with a hydroxyl group in the presence of tetrazole. After adding the oxidizing agent addition, purification was performed by HPLC. A styrene-modified UMP (compound 8) in which the 2 'and 3' positions were phosphorylated was obtained.
[0111] [4]固定工程および SPR金基板上へのタンパク質インプリントポリマーの固定ィ匕 上記 [1 4]で得られた錯体を用いてインプリントポリマーを製造した。 [4] Immobilization Step and Immobilization of Protein Imprinted Polymer on SPR Gold Substrate An imprinted polymer was produced using the complex obtained in [14] above.
[0112] [4 1]金基板上へのアクリル酸の導入 [0112] [4 1] Introduction of acrylic acid onto the gold substrate
N, N，- bis(acryloyl)cystamineの 5 mMメタノール溶液を作製し、これに金基板を浸漬 させることにより重合性官能基であるアクリル酸を基板上に導入した。  A 5 mM methanol solution of N, N, -bis (acryloyl) cystamine was prepared, and a gold substrate was immersed in the solution to introduce acrylic acid, which is a polymerizable functional group, onto the substrate.
[0113] [4— 2]リボヌクレアーゼ 'インプリントポリマーの作製 [0113] [4-2] Ribonuclease 'Preparation of imprinted polymer
リボヌクレアーゼ A(0.33 mM)を铸型タンパク質として用い、上記 [1—4]の銅錯体 C u[cyclen-St]Cl (1.6 mM)とを水中で錯形成させ、 1時間放置して会合させた。その後  Using ribonuclease A (0.33 mM) as a saddle protein, the above [1-4] copper complex Cu [cyclen-St] Cl (1.6 mM) was complexed in water and allowed to associate for 1 hour. . afterwards
2  2
これに、アクリルアミド (1 mM)及び架橋剤メチレンビスアクリルアミド (0.5 mM)を添カロし た。さらに 10 mM Tris緩衝液 (pH7.4)を添加し、これに TEMED 1 mLを加えた。得ら れた溶液に重合開始剤（2,2 ' -ァゾビス [2-メチル - N- 1 , 1-ビス (ヒドロキシメチル) -2-ヒ ドロキシェチル]プロピオンアミド） (50 mM)をカ卩えて均一化させた後、すぐにこの溶液 を上記 [4— 1]で得られた金基板上にキャストした。スライドガラスをかぶせて UV照 射下 1時間重合を行った。重合後、水 'MeOHで洗浄し、さらに Tris緩衝液で洗浄した  This was supplemented with acrylamide (1 mM) and the cross-linking agent methylenebisacrylamide (0.5 mM). Further, 10 mM Tris buffer (pH 7.4) was added, and 1 mL of TEMED was added thereto. Polymerization initiator (2,2'-azobis [2-methyl-N-1,1-bis (hydroxymethyl) -2-hydroxyxetyl] propionamide) (50 mM) (50 mM) was uniformly added to the resulting solution. Immediately after conversion, this solution was cast on the gold substrate obtained in [4-1] above. Polymerization was carried out for 1 hour under UV irradiation with a glass slide. After polymerization, wash with water 'MeOH and further with Tris buffer.
[0114] [4 3] SPR (表面プラズモン共鳴： surface plasmon resonance)センサーを用いたタ ンパク質インプリントポリマーの機能解析 [0114] [4 3] Functional analysis of protein imprinted polymer using SPR (surface plasmon resonance) sensor
5 mM Tris緩衝液 (pH7.4)を流動層とし、タンパク質濃度を 500 mM, 250 mM, 125 mM, 64 mM, 32 mM, 16 mMとして、上記 [4— 2]のインプリントポリマーの SPRセンサ 一としての機能評価を行った。リボヌクレアーゼ Α·インプリントポリマー（上記 [4— 2] のインプリントポリマー）では、 1.1 X 105 Mほどの結合親和性が得られた。 Imprint polymer SPR sensor of [4-2] above with 5 mM Tris buffer (pH 7.4) as fluidized bed and protein concentration of 500 mM, 250 mM, 125 mM, 64 mM, 32 mM, 16 mM. Functional evaluation as one was performed. With the ribonuclease Α imprint polymer (imprint polymer of [4-2] above), a binding affinity of about 1.1 × 10 5 M was obtained.
[0115] [4 4]非インプリントポリマーセンサーとの比較 (図 9) [0115] [4 4] Comparison with non-imprinted polymer sensor (Figure 9)
上記 [4 2]で得られる金基板にっ 、て、タンパク質との複合ィ匕なしに得られるポリ マー薄膜を非インプリントポリマーと呼ぶ。これについても同様に調製を行うことがで きる。上記 [4— 2]で得られたインプリントポリマーおよび、非インプリントポリマーにつ いて、 10 mM Tris緩衝液（pH7. 4)を流動層とし、 25°C、 [リボヌクレアーゼ A] = 0.25 mMの条件で、 SPRセンサーとしての機能を比較した。結果を図 9に示す。その結 果、インプリンティングを行った方がリボヌクレアーゼ Aに対しては結合能が高 、ことが 分力つた。さらに、金属を再度添加しもとの錯体に戻すことにより、結合量を再度もと の状態に戻すことも可能であった（図 15)。 The polymer thin film obtained without the complex with protein by the gold substrate obtained in [42] above is called a non-imprinted polymer. This can be done in the same way. wear. For the imprinted polymer and non-imprinted polymer obtained in [4-2] above, use 10 mM Tris buffer (pH 7.4) as a fluidized bed, 25 ° C, and [ribonuclease A] = 0.25 mM. The function as an SPR sensor was compared under certain conditions. The results are shown in FIG. As a result, it was found that the imprinting had higher binding ability to ribonuclease A. Furthermore, by adding the metal again and returning it to the original complex, it was also possible to return the binding amount to the original state again (Fig. 15).
[0116] [4— 5]エチレンジァミンを機能性モノマーとして用いた場合のインプリントポリマー センサーの吸着活性 (図 16)  [0116] [4-5] Adsorption activity of imprinted polymer sensor using ethylenediamine as a functional monomer (Figure 16)
リボヌクレアーゼ Aをテンプレートとし、エチレンジァミンを機能性モノマーとして利 用し、 GEMAを添加モノマーとして合成した薄膜についての相対吸着量を検討したと ころ、吸着量について約 2倍ほどの上昇が見られた。この機能性ポリマーについては 、塩基性タンパク質に対して吸着活性の上昇が見られた。  When the relative adsorption amount of a thin film synthesized using ribonuclease A as a template, ethylenediamine as a functional monomer, and GEMA as an additive monomer was examined, the adsorption amount increased by about 2 times. For this functional polymer, an increase in adsorption activity was observed for basic proteins.
[0117] [4— 6]サイクレンを機能性モノマーとして用いた場合のインプリントポリマーセンサ 一の吸着活性 (図 17)  [0117] [4-6] Imprinted polymer sensor using cyclone as a functional monomer One adsorption activity (Fig. 17)
リボヌクレアーゼ Aをテンプレートとし、エチレンジァミンを機能性モノマーとして利 用し、 GEMAを添加モノマーとして合成した薄膜についての相対吸着量を検討したと ころ、やはり上記と同様に吸着率の上昇が見られた。この機能性ポリマーについては 、ターゲットに対する吸着率の選択性を有した。  When the relative adsorption amount of a thin film synthesized using ribonuclease A as a template, ethylenediamine as a functional monomer, and GEMA as an additive monomer was examined, an increase in the adsorption rate was also observed. This functional polymer had selectivity of the adsorption rate with respect to the target.
[0118] [4— 7]エチレンジァミンを機能性モノマーとして用いた場合のインプリントポリマー センサーの塩強度依存性 (図 18)  [0118] [4-7] Dependence of salt strength of imprinted polymer sensor using ethylenediamine as a functional monomer (Fig. 18)
リボヌクレアーゼ Aをテンプレートとし、エチレンジァミンを機能性モノマーとして利 用し、 GEMAを添加モノマーとして合成した薄膜についての相対吸着量を検討したと ころ、塩強度が上昇するに従って吸着率は低減した。また、 GEMAだけではほとんど タンパク質は吸着せず、非特異的吸着の低減に有効であることが分力つた。  When the relative adsorption amount of a thin film synthesized using ribonuclease A as a template, ethylenediamine as a functional monomer, and GEMA as an additive monomer was examined, the adsorption rate decreased as the salt strength increased. In addition, it was found that GEMA alone hardly adsorbs proteins and is effective in reducing nonspecific adsorption.
[0119] [4— 8]ポルフィリンとリボヌクレアーゼ Aとの相互作用解析 (図 19)  [0119] Analysis of interaction between [4-8] porphyrin and ribonuclease A (Fig. 19)
ビアコア社製 CM- 5基盤に固定化したリボヌクレアーゼ Aに対する TCPP(Tetrakis(4 -carboxyphenyDporphine),及び Zn(TCPP)の親和性を検討した。センサーグラムから 親和性を検討したところ、 pH9において、約 30000M—1ほどの結合定数が得られた [0120] [4 9]ポルフィリン（1^^?(丁61^ 3(4-0&1¾0 1½ 1 0卬1^½))とリボヌクレァーゼ Aとの相互作用解析 (図 20) We investigated the affinity of TCPP (Tetrakis (4-carboxyphenyDporphine) and Zn (TCPP) to ribonuclease A immobilized on Biacore's CM-5 base. — A coupling constant of about 1 was obtained [0120] [4 9] Interaction analysis between porphyrin (1 ^^? (Chi 61 ^ 3 (4-0 & 1¾0 1½ 1 0 卬 1 ^ ½)) and ribonuclease A (Fig. 20)
UVスペクトルを用いてリボヌクレアーゼ Aと Zn(TCPP)との親和性を検討した。滴定 曲線力も得られたフィッティング力も親和性を検討したところ、 pH9において、約 250 OOM—1ほどの結合定数が得られた（[タンパク質] = [リボヌクレアーゼ A] = 5 mM,トリ ス緩衝液、 ρΗ9)。この結果は [4 8]の結果と一致している。 The affinity between ribonuclease A and Zn (TCPP) was examined using UV spectra. As a result of examining the affinity of both the titration curve force and the obtained fitting force, a binding constant of about 250 OOM- 1 was obtained at pH 9 ([protein] = [ribonuclease A] = 5 mM, Tris buffer, ρΗ9 ). This result is consistent with the result of [48].
[0121] [4— 10]ポルフィリンを機能性モノマーとして用いた場合のインプリントポリマーセン サ一の各タンパク質に対する親和性 (図 21) [0121] [4-10] Affinity of imprint polymer sensor for each protein when using porphyrin as a functional monomer (Fig. 21)
リボヌクレアーゼ Aをテンプレートとし、ビュル修飾した Zn(TCPP)を機能性モノマー として利用して合成した薄膜についての相対吸着量を検討した。その結果、テンプレ ートであるリボヌクレアーゼ Aに対する親和性が著しく向上し、 目的タンパク質に対す るセンサーとして有用であることが分力つた。  We investigated the relative adsorption of thin films synthesized using ribonuclease A as a template and bully-modified Zn (TCPP) as a functional monomer. As a result, the affinity for ribonuclease A, a template, was remarkably improved, and it was found useful as a sensor for the target protein.
[0122] [5]基質阻害剤を用いたインプリンティング [0122] [5] Imprinting using substrate inhibitors
上記 [3]で合成した 2，一UMPスチレンを機能性モノマーとして用い、リボヌクレア一 ゼのインプリンティングを行った。 2，一UMPスチレンをモノマーとして用いて検討した 結果、固定ィ匕することにより、均一系における親和性よりも 10倍ほどの結合親和性が 得られた。さら〖こ、インプリンティングを行うことにより、飽和結合量と親和性の向上が 見られた。  Ribonuclease was imprinted using 2,1 UMP styrene synthesized in [3] above as a functional monomer. As a result of studies using UMP styrene as a monomer, a binding affinity of about 10 times that of a homogeneous system was obtained by immobilization. Furthermore, the amount of saturated binding and affinity were improved by imprinting.
[0123] [6]蛍光色素修飾モノマーを用いたセンシング  [0123] [6] Sensing using fluorescent dye-modified monomers
ダンシル修飾銅エチレンジァミン錯体を機能性モノマーとして用い、メチレンビスァ クリルアミドを架橋剤、アクリルアミドをコモノマーとしてラジカル重合を行った。テンプ レートタンパク質の除去を行った後、蛍光プレートリーダーによる分析を行った。その 結果、このインプリントポリマーでは、タンパク質の添カ卩に伴う短波長シフトと蛍光強 度の増大が見られた。このことは、本インプリントポリマーのプロテインチップへの応用 が可能なことを示している。  Radical polymerization was performed using dansyl-modified copper ethylenediamine complex as a functional monomer, methylenebisacrylamide as a crosslinking agent, and acrylamide as a comonomer. After removing the template protein, analysis with a fluorescent plate reader was performed. As a result, in this imprinted polymer, a short wavelength shift and an increase in fluorescence intensity were observed with protein addition. This indicates that the imprint polymer can be applied to protein chips.
[0124] [7]配位子の添カ卩による基質特異性の向上 [0124] [7] Improvement of substrate specificity by ligand addition
铸型タンパク質であるリボヌクレアーゼ Aに対して 5等量のエチレンジァミンー銅錯 体を複合ィ匕させた。これと機能性モノマーであるアクリルアミド、及びメチレンビスァク リルアミドを添加した後、複合ィ匕して ヽな 、金属配位を消滅させるためにエチレンジ アミンを 2等量添加した。これを重合して機能性モノマーを固定ィ匕したところ、得られ たポリマーはリボヌクレアーゼ Aに対する高い選択性を得ることができた。 5 equivalents of ethylenediamine-copper complex with ribonuclease A I made my body complex. After addition of this and the functional monomers acrylamide and methylenebisacrylamide, 2 equivalents of ethylenediamine were added in order to eliminate the metal coordination. When this was polymerized to immobilize functional monomers, the resulting polymer was able to obtain high selectivity for ribonuclease A.
[0125] [8]タンパク質の定量および同定 [0125] [8] Protein quantification and identification
上記で作製したインプリントポリマーを用いた以外は、下記〔II〕の [1 2]と同様の 操作を行った。その結果、下記〔II〕の [1— 3]と同様に、タンパク質の定量および同 定が可能であることが分力つた (データ不図示)。  The same operation as [12] in [II] below was performed except that the imprinted polymer prepared above was used. As a result, in the same way as [1-3] in [II] below, it was possible to quantify and identify proteins (data not shown).
[0126] [9]タンパク質のリフォールディング [0126] [9] Protein refolding
上記で作製したインプリントポリマーを用いた以外は、下記〔II〕の [2]と同様の操作 を行った。その結果、下記〔II〕の [2— 5]と同様に、リゾチームのリフォールデイングが 促進された。  The same operation as [2] in [II] below was carried out except that the imprinted polymer prepared above was used. As a result, lysozyme refolding was promoted in the same way as [2-5] in [II] below.
[0127] 〔II〕インプリントポリマー [II] Imprint polymer
[1]インプリントポリマーの作製および評価  [1] Fabrication and evaluation of imprinted polymer
[1 - 1]インプリントポリマーの合成  Synthesis of [1-1] imprinted polymer
4種の铸型タンパク質（リゾチーム（LY)、シトクロム C (CY)、リボヌクレアーゼ A (RN) 、ラクトアルブミン (LA) )、および 2種の機能性ポリマー (銅一エチレンジァミン (Cu(en) )、銅一サイクレン (Cu(cyc》を組み合わせて、 8種のインプリントポリマー LY- Cu(en)IP, LY-Cu(cyc)IP, CY— Cu(en)IP, CY— Cu(cyc)IP, RN— Cu(en)IP, RN— Cu(cyc)IP, LA— Cu( en)IP, LA-Cu(cyc)IP,および 2種類の非インプリントポリマー（Cu(en)- NIP, Cu(en)- NI P)を作製した。  Four types of saddle proteins (lysozyme (LY), cytochrome C (CY), ribonuclease A (RN), lactalbumin (LA)), and two functional polymers (copper-ethylenediamine (Cu (en)), copper One cyclen (Cu (cyc) combined with 8 types of imprinted polymers LY-Cu (en) IP, LY-Cu (cyc) IP, CY—Cu (en) IP, CY—Cu (cyc) IP, RN) — Cu (en) IP, RN— Cu (cyc) IP, LA— Cu (en) IP, LA-Cu (cyc) IP, and two non-imprinted polymers (Cu (en) -NIP, Cu (en )-NI P) was produced.
[0128] インプリントポリマーの名称の頭の部分は铸型タンパク質を示し、その次に記す Cu( en)は用いた機能性モノマーを示す。  [0128] The head portion of the name of the imprint polymer indicates a cage protein, and Cu (en) described next indicates the functional monomer used.
[0129] 非インプリントポリマーは、铸型タンパク質を重合時に添加しな力つた以外は、イン プリントポリマーと同様の操作によって作製した。非インプリントポリマーの名称の前半 は、用いた機能性ポリマーを指し、名称の最後に NIPを付して、 IPと区別する。具体的 な作製は、表 1に示すレシピに沿って行った。トリス緩衝液 (TB、 50 mM, pH7.4) 15 mL中に、表 1に示すポリマー毎の規定濃度になるように Cu(en)もしくは Cu(cyc)を溶解 させ、 pHを 7.4に合わせた後、溶液を 20 mLにメスアップした（プレポリマーミックス）。 バイアル瓶にプレポリマーミックス 7.2 mLを取り、そこへ表 1に示す量の GEMA:グル コシロキシェチルメタタリレート、 MBAA:N, N，-メチレンビスアクリルアミド、および標 的タンパク質を溶解させ、そのまま 30分攪拌した (ポリマーミックス)。ポリマーミックス の窒素置換を 5分間行い、重合開始剤 (VA-80)を加えて速やかに攪拌し、バイアル 瓶をパラフィルムで密封して UV照射下 4°Cで 18時間重合を行った。シトクロム Cを铸 型分子として用いたポリマーミックスについては、光重合によって固まらな力つたので 、重合開始剤である N, N, Ν' , Ν' -テトラメチルエチレンジァミン (TEMED)、過硫酸 アンモニゥム (APS)による重合を行った。 [0129] The non-imprinted polymer was produced in the same manner as the imprinted polymer, except that the anchor protein was not added during polymerization. The first half of the name of the non-imprinted polymer refers to the functional polymer used and is distinguished from IP by adding NIP at the end of the name. The specific production was performed according to the recipe shown in Table 1. Dissolve Cu (en) or Cu (cyc) in 15 mL of Tris buffer (TB, 50 mM, pH 7.4) to the specified concentration for each polymer shown in Table 1. After adjusting the pH to 7.4, the solution was diluted to 20 mL (prepolymer mix). Take 7.2 mL of the prepolymer mix in a vial and dissolve the amounts of GEMA: glycosylchetylmetatalylate, MBAA: N, N, -methylenebisacrylamide, and the target protein shown in Table 1 into the vial. Stir for 30 minutes (polymer mix). The polymer mix was purged with nitrogen for 5 minutes, a polymerization initiator (VA-80) was added and stirred rapidly, the vial was sealed with parafilm, and polymerized at 4 ° C for 18 hours under UV irradiation. For polymer mixes using cytochrome C as a cage molecule, photopolymerization gave a solid force, so the polymerization initiators N, N, Ν ', Ν' -tetramethylethylenediamine (TEMED), persulfate Polymerization with ammonia (APS) was carried out.
[0130] [表 1] [0130] [Table 1]
IP NIP IP NIP
標的タンパク質 フ.5 μτηοΐ - 機能性モノマー 0.15 mmol  Target protein F.5 μτηοΐ-Functional monomer 0.15 mmol
GEMA 394.2 mg(l .35 lnmol)  GEMA 394.2 mg (l .35 lnmol)
MBAA 1.5ιτιιτιο1 231 mg)  MBAA 1.5ιτιιτιο1 231 mg)
VA-080 24.フ mol (lO.l mg)  VA-080 24 mol (lO.l mg)
ΤΒ (50ιτιΜ,ρΗ7.4) 7.2 mL '  ΤΒ (50ιτιΜ, ρΗ7.4) 7.2 mL '
得られたインプリントポリマーは、スパチェラで軽く砕き、 0.5 M NaCl水溶液、水で洗 浄したのち、 5 %酢酸で一晩攪拌することによって洗浄を行った。その後、水、 1 M H C1もしくは 1 M NaOH水溶液、水の順に洗浄を行った。ポリマー力もの铸型タンパク質 の洗浄量は、可視紫外分光光度計を用いて洗液の 280 nmにおける吸光度を測定す ることによって求めた。洗浄したポリマーは、 24時間凍結乾燥を行った。 The obtained imprinted polymer was lightly crushed with a spatula, washed with 0.5 M NaCl aqueous solution and water, and then washed with 5% acetic acid overnight. Then, it wash | cleaned in order of water, 1 MHC1 or 1 M NaOH aqueous solution, and water. The amount of washing of the protein-type saddle protein was determined by measuring the absorbance of the washing solution at 280 nm using a visible ultraviolet spectrophotometer. The washed polymer was lyophilized for 24 hours.
[0131] なお、下記 [1 2]の再結合実験に用いるのに十分な 90%以上の洗浄率が得られ たことを確認した。 [0131] It was confirmed that a washing rate of 90% or more sufficient for use in the recombination experiment of [12] below was obtained.
[0132] [1— 2] 6種類のタンパク質（ミオグロビン、リボヌクレア一ゼ八、リゾチーム、アルブミ ン、シトクロム C、ラクトアルブミン）を用いた再結合実験 1) 4種類のタンパク質混合物を用いた再結合実験 [0132] [1-2] Recombination experiments using 6 types of proteins (myoglobin, ribonuclease 1-8, lysozyme, albumin, cytochrome C, lactalbumin) 1) Recombination experiments using 4 protein mixtures
インプリントポリマー、非インプリントポリマーそれぞれ 5 mgをバイアル瓶に量り取り、 そこへタンパク質溶液 (リゾチーム，リボヌクレアーゼ A,ミオグロビン，アルブミン， 0.25 mg/mL, 5 mL, in TB (50 mM, pH7.4》を分注し、バイアルを密封し、ローターで攪拌 させながら、 16時間、 15°Cの条件でインキュベーションを行った。シリンジフィルター (DISMIC-25CS, ADVANTEC)を用いてポリマーをろ過し、上澄みの濃度を HPLCに よって定量し、ポリマーへの铸型タンパク質の結合量を求めた。  Weigh 5 mg each of imprinted polymer and non-imprinted polymer in a vial, and put protein solution (lysozyme, ribonuclease A, myoglobin, albumin, 0.25 mg / mL, 5 mL, in TB (50 mM, pH7.4)) The vial was sealed and stirred with a rotor, and incubated for 16 hours at 15 ° C. The polymer was filtered using a syringe filter (DISMIC-25CS, ADVANTEC), and the concentration of the supernatant was determined. Was quantified by HPLC, and the amount of the anchor protein bound to the polymer was determined.
[0133] HPLC条件は以下の通りである。 [0133] HPLC conditions are as follows.
カラム： TSKgel Butyト NPR 4.6 mm ID X 3.5 cm (東ソ一）  Column: TSKgel Buty NPR 4.6 mm ID X 3.5 cm (Tosohichi)
溶離液; A: 0.1 Mトリス緩衝液 (pH7.0)  Eluent; A: 0.1 M Tris buffer (pH 7.0)
B : 0.1 Mトリスナトリウム緩衝液 (pH7.0) + 2.5 M硫酸アンモ-ゥム  B: 0.1 M Tris sodium buffer (pH 7.0) + 2.5 M ammonium sulfate
A (100 %)→B (25 %) リニアグラジェント（9 min)  A (100%) → B (25%) Linear gradient (9 min)
2)シトクロム C、ラクトアルブミンを用いた再結合実験  2) Rebinding experiment using cytochrome C and lactalbumin
インプリントポリマーの铸型として用いた 2種類のタンパク質シトクロム C、ラクトアル ブミンはタンパク質混合物の中に加えても、先に用いた TSKgel Buty卜 NPRによる迅 速分離が不可能であったため、これらのサンプルは個別にポリマーと再結合実験を 行った。  The two types of proteins cytochrome C and lactalbumine used as the imprint polymer cage were not able to be separated quickly by the previously used TSKgel ButyPR NPR, even though they were added to the protein mixture. Conducted recombination experiments with the polymer individually.
[0134] インプリントポリマー、非インプリントポリマーそれぞれ 5 mgをバイアル瓶に量り取り、 そこへタンパク質溶液 (シトクロム Cもしくはラクトアルブミン， 0.25 mg/mL, 5 mL, in TB (50 mM, pH7.4》を分注し、バイアルを密封し、ローターで攪拌させながら、 16時間 、 15°Cの条件でインキュベーションを行った。シリンジフィルター（DISMIC-25CS, AD -VANTEC)を用いてポリマーをろ過し、上澄みの濃度を HPLCによって定量し、ポリ マーへの铸型タンパク質の結合量を求めた。  [0134] 5 mg each of imprinted polymer and non-imprinted polymer is weighed into a vial, and there a protein solution (cytochrome C or lactalbumin, 0.25 mg / mL, 5 mL, in TB (50 mM, pH7.4) The vial was sealed, and the mixture was stirred for 16 hours at 15 ° C with stirring on a rotor.The polymer was filtered using a syringe filter (DISMIC-25CS, AD-VANTEC), and the supernatant was filtered. The concentration of the protein was quantified by HPLC, and the amount of the anchor protein bound to the polymer was determined.
[0135] HPLC条件条件は以下の通りである。  [0135] The HPLC conditions are as follows.
カラム： TSKgel G-3000SWXL 7.8 mm X 30 cm (東ソ一）  Column: TSKgel G-3000SWXL 7.8 mm X 30 cm (Tosohichi)
溶離液； A: 0.05 Mトリス緩衝液 (ρΗ7.0) +0.3 M NaCl  Eluent: A: 0.05 M Tris buffer (ρΗ7.0) +0.3 M NaCl
[1 3]結果  [1 3] Results
図 10に、疎水性クロマトグラフィーを用いたタンパク質分離のクロマトグラムを示す。 塩濃度リニアグラジェントにより、 8分間程度の短時間で 4種類のタンパク質が完全に 分離されて ヽることがゎカゝる。 Figure 10 shows a chromatogram of protein separation using hydrophobic chromatography. The salt concentration linear gradient allows the four proteins to be completely separated in as little as 8 minutes.
[0136] 銅 エチレンジァミン (Cu(en))を機能性モノマーに用 、たポリマー群および銅 サ イクレン (Cu(cyc))を機能性モノマーに用いたポリマー群に対するタンパク質混合物 の結合挙動データに対してそれぞれ主成分分析を行った。結果を図 11及び図 12〖こ 示す。実験は繰り返し 5回行ったため、プロットはそれぞれのタンパク質について 5つ ある。 [0136] For the binding behavior data of a protein mixture to a polymer group using copper ethylenediamine (Cu (en)) as a functional monomer and a polymer group using copper cyclene (Cu (cyc)) as a functional monomer A principal component analysis was performed for each. The results are shown in Fig. 11 and Fig. 12. Since the experiment was repeated five times, there are five plots for each protein.
[0137] 各ポリマーを 1つのセンサーチャネルとしてとらえ、おのおの 5チャネルからなるセン サ一による出力と考えられる。同じタンパク質の 5つのデータプロットは同様の重みで 互いに 1つのクラスターを形成していることが分かる。そして、各クラスタ一はお互いが 重なることなく全く独立していた。また、用いた二種類の機能性モノマーによって、や や異なるクラスターの分布が見られた。  [0137] Each polymer is considered as one sensor channel, and it is thought to be output from a sensor consisting of 5 channels each. It can be seen that the five data plots of the same protein form one cluster with the same weight. Each cluster was completely independent without overlapping each other. A slightly different cluster distribution was observed depending on the two types of functional monomers used.
[0138] 以上、複数のインプリントポリマーを用いて、実サンプルを志向した混合物を用いた 場合、各ポリマーは各タンパク質に対して特徴的なレスポンスを示し、主成分分析を 行うことで各タンパク質のデータは完全に独立した 4つのクラスターに分離された。そ れらにカ卩えて、シトクロム C,ラクトアルブミンについても、それぞれ独立したクラスター を形成していた。そして、計 6つのクラスターに分離され、タンパク質混合物中の成分 の同定が本法を用いることで可能なことが示された。  [0138] As described above, when a mixture intended for an actual sample is used by using a plurality of imprinted polymers, each polymer exhibits a characteristic response to each protein, and a principal component analysis is performed to analyze each protein. The data was separated into four completely independent clusters. In addition to these, cytochrome C and lactalbumin also formed independent clusters. And it was separated into a total of 6 clusters, and it was shown that the identification of the component in the protein mixture was possible by using this method.
[0139] [2]インプリントポリマーを人工分子シャペロンとして用いた変性リゾチームのリフォ 一ノレディング  [0139] [2] Modification of modified lysozyme using imprinted polymer as artificial molecular chaperone
[2— 1]インプリントポリマーの作製  [2-1] Preparation of imprinted polymer
リゾチームインプリントポリマーおよび非インプリントポリマーは、上記 [1 1]の LY-C u(en)IP、 Cu(en)- NIPとおなじものを合成した。これら 2つのポリマーを後述の変性リゾ チーム再生実験で用いた。  The lysozyme imprinted polymer and non-imprinted polymer were synthesized in the same manner as LY-Cu (en) IP and Cu (en) -NIP in [11] above. These two polymers were used in the modified lysozyme regeneration experiment described below.
[0140] [2— 2]リゾチームの変性 [0140] [2-2] Denaturation of lysozyme
変性用緩衝液（8M 尿素、 ImM EDTA、 10mM DTT、 0. ImM Tris—HC L、 pH8. 5)中にリゾチームを 10.0 mg/mLの濃度になるように溶解させ、 38°Cで 2.5 時間攪拌し、変性を行った。変性後の液を以下、変性液と称する。変性の確認は、下 記 [2— 3]の酵素活性測定によって行い、その活性が 2 %以下であることを確認した。 Dissolve lysozyme to a concentration of 10.0 mg / mL in denaturing buffer (8M urea, ImM EDTA, 10 mM DTT, 0. ImM Tris—HCl, pH 8.5) and stir at 38 ° C for 2.5 hours Then, denaturation was performed. Hereinafter, the modified solution is referred to as a modified solution. Check for denaturation below. It was confirmed by the enzyme activity measurement described in [2-3] and that the activity was 2% or less.
[0141] [2— 3]酵素活性測定  [0141] [2-3] Enzyme activity measurement
リゾチームの基質 (Micrococcus lysodeikticus (乾燥細胞壁》を 50 mMトリス緩衝液（ pH 6.2)〖こ 0.25 mg/mLになるように懸濁させ、ー晚撹拌したものを基質溶液として調 製した。この懸濁液 3 mLを恒温セル（35°C—定）に取り、リゾチーム溶液 (9 μ L)をカロ えて混合し、可視紫外分光光度計を用いて、 450 nmにおける吸光度の減少を測定し た。吸光度の減少の測定開始時間における傾き (酵素反応の初速度)からリゾチーム の酵素活性を求めた。測定するリゾチーム溶液は、活性が濃度に比例する範囲内の 濃度になるように 50 mMトリス緩衝液で希釈して用いた。なお、懸濁液 3 mLとリゾチ ーム溶液 9 Lをセル内で混合した時に 1分間に 1 Absの吸光度の減少を与える酵 素量を 1 Uと定義して、リゾチーム濃度とその酵素活性の関係をプロットした検量線を 作成した (不図示)。  A lysozyme substrate (Micrococcus lysodeikticus (dried cell wall)) was suspended in 50 mM Tris buffer (pH 6.2) at a concentration of 0.25 mg / mL. 3 mL of the solution was placed in a constant-temperature cell (35 ° C-constant), mixed with lysozyme solution (9 μL), and the decrease in absorbance at 450 nm was measured using a visible ultraviolet spectrophotometer. The enzyme activity of lysozyme was determined from the slope of the measurement start time (initial rate of enzyme reaction), and the lysozyme solution to be measured was diluted with 50 mM Tris buffer so that the activity was in a range proportional to the concentration. The amount of enzyme that gives a decrease in absorbance of 1 Abs per minute when 3 mL of the suspension and 9 L of lysozyme solution are mixed in the cell is defined as 1 U, and lysozyme Create a calibration curve plotting the relationship between concentration and enzyme activity (Not shown).
[0142] [2— 4]変性リゾチームの再活性ィ匕  [0142] [2-4] Reactivation of modified lysozyme
以下に示す 1)および 2)の 2つの手法を用いて、ポリマーを用いた変性リゾチームの 再活性化実験を行った。  Using the two methods 1) and 2) shown below, a reactivation experiment of modified lysozyme using a polymer was conducted.
[0143] 1) batch型（グルタチオン添加）  [0143] 1) Batch type (with glutathione added)
変性液 3 mLを再生用緩衝液 (1.5 M Urea, 3 mM Glutathione reduced form, 5 mM Glutathione oxidized form, 1 mM EDTA, 0.1 M Tris— HC1, pH8.0)27 mLと混合し、 10 倍に希釈した。希釈溶液は 3つの容器にそれぞれ 30 mLずつ用意した。  3 mL of denaturing solution is mixed with 27 mL of regeneration buffer (1.5 M Urea, 3 mM Glutathione reduced form, 5 mM Glutathione oxidized form, 1 mM EDTA, 0.1 M Tris—HC1, pH 8.0) and diluted 10-fold did. 30 mL each of the diluted solution was prepared in three containers.
[0144] 3つの容器のうちの一つ、すなわちポリマーをカ卩えないもの（NP)については希釈後 すぐに溶液の吸光度を計り、これを初濃度とした。また、残りの 2つには LY-Cu(en)IP 、または Cu(cyc)- NIPをそれぞれ 60 mgずつを加えた。  [0144] For one of the three containers, that is, one that does not contain polymer (NP), the absorbance of the solution was measured immediately after dilution, and this was used as the initial concentration. In addition, 60 mg each of LY-Cu (en) IP or Cu (cyc) -NIP was added to the remaining two.
[0145] 以上のような 3つの溶液を室温で攪拌し、タンパク質の再生を行った。再生液で希 釈してから 0.5, 1, 2, 3, 4, 5, 7, 12, 24, 27時間経過した時点で反応容器からリゾチ一 ム溶液を 2 mL採取した。ポリマーをカ卩えたものについてはシリンジフィルター（DISMI C-25CS, ADVANTEC)でポリマーをろ過し、随時、吸光度 (280 nm)を測定し、溶液中 のリゾチーム濃度を求めた。また、先述の手法で採取した溶液の酵素活性を測定し た。上記 [2— 3]で求めた溶液の酵素活性を検量線によって、リフォールデイング済 みのリゾチームの量に換算した。リフォールディング率は同濃度の天然リゾチームと の酵素活性の比で表した。 [0145] The three solutions as described above were stirred at room temperature to regenerate proteins. At 0.5, 1, 2, 3, 4, 5, 7, 12, 24, and 27 hours after dilution with the regenerating solution, 2 mL of the lysozyme solution was collected from the reaction vessel. For those with a polymer, the polymer was filtered with a syringe filter (DISMI C-25CS, ADVANTEC), and the absorbance (280 nm) was measured as needed to determine the lysozyme concentration in the solution. In addition, the enzyme activity of the solution collected by the method described above was measured. The enzyme activity of the solution obtained in [2-3] above has been refolded using a calibration curve. Converted to the amount of lysozyme. The refolding rate was expressed as a ratio of enzyme activity to natural lysozyme at the same concentration.
[0146] 2) Flow型（グルタチオン未添加）  [0146] 2) Flow type (no glutathione added)
1.5 gの LY- Cu(en)IP、または Cu(en)- NIPをそれぞれ充填した固相合成用チューブ に、変性液 0.3 mLを注ぎ、これらポリマーにリゾチームを吸着させた。次に、ダルタチ オンを含まない再生用緩衝液 (1.5 M Urea, 1 mM EDTA, 0.1 M Tris- HC1, pH8.0)2. 7 mLを 3回に分けて、固定相に注いだ。その後、吸着したタンパク質を 50 mMクェン 酸-タエン酸ナトリウム緩衝液 (pH6.0)で溶出させ、一回目は 3 mL,その後は 2 mLごと に計 15 mLのフラクションを取った。各フラクションの吸光度を測定し、初期量をほぼ 1 00 %回収したことを確認した。  0.3 mL of the denaturing solution was poured into solid-phase synthesis tubes filled with 1.5 g of LY-Cu (en) IP or Cu (en) -NIP, respectively, to adsorb lysozyme to these polymers. Next, 2.7 mL of regeneration buffer (1.5 M Urea, 1 mM EDTA, 0.1 M Tris-HC1, pH 8.0) not containing dartathione was poured into the stationary phase in three portions. Thereafter, the adsorbed protein was eluted with 50 mM citrate-sodium taenoate buffer (pH 6.0), and 3 mL was collected at the first time, and then 15 mL fractions were taken every 2 mL. The absorbance of each fraction was measured, and it was confirmed that an initial amount of approximately 100% was recovered.
[0147] 各フラクションを全て合わせた溶液の酵素活性を、上記 1)と同様の方法で測定した 。また、リファレンスとして 0.3 mLの変性液に 2.7 mLの再生液を、ポリマーを入れたも のと同様の時間を要して滴下したものを調整し、その酵素活性も測定した。酵素活性 測定は、基質溶液 3000 μ Lに対してリゾチームを含む全フラクション 90 μ Lを反応さ せ、以下前述の方法と同様に行った。求めた酵素活性から、リゾチームのリフォール ディング率を求めた。  [0147] The enzyme activity of the solution in which all the fractions were combined was measured by the same method as in 1) above. In addition, as a reference, 2.7 mL of a regenerating solution was added dropwise to 0.3 mL of a denatured solution over the same time as the polymer was added, and the enzyme activity was also measured. The enzyme activity was measured by reacting 90 μL of the total fraction containing lysozyme with 3000 μL of the substrate solution, and performing the same procedure as described above. The refolding rate of lysozyme was determined from the determined enzyme activity.
[0148] [2— 5]結果  [0148] [2—5] Results
1) batch型の結果 LY- Cu(en)IP、または Cu(en)- NIP  1) Batch type result LY-Cu (en) IP or Cu (en) -NIP
図 13にインプリントポリマー添カ卩時におけるリゾチームのリフォールデイング率の経 時変化を示す。なお、 24, 27時間後のリフォールディング率は約 90%であり、これ は 8時間経過の段階とほぼ同様であった。そのため、リフォールディング率は約 8時 間でほぼ頭打ちになると考えられる。  Figure 13 shows the changes over time in the lysozyme refolding rate when the imprinted polymer was added. The refolding rate after 24 and 27 hours was about 90%, which was almost the same as the 8 hour stage. As a result, the refolding rate is expected to reach its peak at about 8 hours.
[0149] 図 13に示すように、 batch型の再生系では、特に 3, 4時間といった反応初期段階で は、ポリマーを添カ卩した時のリフォールデイング率力 添カ卩しなかったものよりも高かつ た。また、 Cu(en)- NIPより LY- Cu(en)IPのほうがその効果が大きいことから、本実験で 合成した LY-Cu(en)IPは铸型に基づく分子シャペロンとしての機能を有していることが 分かった。  [0149] As shown in Fig. 13, in the batch-type regeneration system, especially at the initial reaction stage such as 3 or 4 hours, the refolding rate when the polymer was added was higher than that without the addition. It was too expensive. In addition, LY-Cu (en) IP has a greater effect than Cu (en) -NIP, so LY-Cu (en) IP synthesized in this experiment has a function as a molecular chaperone based on the saddle type. I found out.
[0150] 2) Flow型の結果 図 14に、上記 [2— 4]の 2)における溶出量と吸光度の関係（クロマトグラム）を示す 。図 14より、リゾチームの溶出に要する液量は Cu(en)- NIPよりも LY- Cu(en)IPのほう が多いことが分かる。これは、インプリントポリマーのほうがリゾチームの保持能が高い ためであると考えられる。 [0150] 2) Result of Flow type Fig. 14 shows the relationship (chromatogram) between the elution amount and absorbance in [2-4] 2) above. From Fig. 14, it can be seen that the amount of liquid required for elution of lysozyme is greater for LY-Cu (en) IP than for Cu (en) -NIP. This is thought to be because the imprint polymer has higher lysozyme retention.
[0151] リフォールデイング率については、上記 1)の batch型の結果と同様に、インプリントポ リマーによってリフォールデイング率が高かった（データ不図示）。すなわち、 LY- Cu( en)IPは分子シャペロンとして機能して 、た。  [0151] Regarding the refolding rate, the refolding rate was high due to the imprint polymer (data not shown), similar to the batch type result in 1) above. That is, LY-Cu (en) IP functioned as a molecular chaperone.
[0152] 〔III〕タンパク質の固定ィ匕方法  [0152] [III] Protein immobilization method
2，- CMPを pH5. 5の酢酸緩衝液中でリボヌクレアーゼ A 300 Mに対して 1当量 添加し、複合化させた。これに銅—サイクレン錯体を 5等量添加し、会合させた。これ にアクリルアミド 3等量、メチレンビスアクリルアミド 1.5当量を添加し、ラジカル開始剤 過硫酸カリウム、 TEMEDを添加し、ラジカル重合を行った。その後 pHを 7. 4として 2， — CMPを洗浄により除去し、基質結合部位がブロックされていない固定化リボヌタレ ァーゼを得た。得られた固定ィ匕タンパク質は、 2'—CMPを添加せずに固定ィ匕したタ ンパク質と比較すると、もとの活性を失うことなく固定化されていることが分力 た。  2, -CMP was added in an equivalent amount of pH 5.5 to 1 ribonuclease A 300 M and complexed. To this was added 5 equivalents of a copper-cyclene complex and allowed to associate. To this was added 3 equivalents of acrylamide and 1.5 equivalents of methylenebisacrylamide, and radical initiators potassium persulfate and TEMED were added to perform radical polymerization. After that, the pH was adjusted to 7.4 and 2, — CMP was removed by washing to obtain an immobilized ribonuclease whose substrate binding site was not blocked. It was found that the obtained immobilized protein was immobilized without losing its original activity, compared with the protein immobilized without adding 2'-CMP.
[0153] 尚、発明を実施するための最良の形態の項においてなした具体的な実施態様また は実施例は、あくまでも、本発明の技術内容を明らかにするものであって、そのような 具体例にのみ限定して狭義に解釈されるべきものではなぐ本発明の精神と次に記 載する特許請求の範囲内で、いろいろと変更して実施することができるものである。 産業上の利用の可能性  It should be noted that the specific embodiments or examples made in the section of the best mode for carrying out the invention are merely to clarify the technical contents of the present invention, and Various modifications may be made within the spirit of the present invention and the following claims, which should not be construed as narrowly limited to only examples. Industrial applicability
[0154] 本発明のインプリントポリマーは、ライフサイエンス、食品分野、環境分野、バイオテ クノロジ一におけるタンパク質の同定や単離のためのクロマトグラフィー等の分離剤、 診断のための試薬やプロテインチップ、バイオテクノロジーを用いて人工的に生産し たタンパク質の同定ゃリフォールデイング工程等に利用可能である。 [0154] The imprinted polymer of the present invention is a separation agent such as chromatography for identification and isolation of proteins in life science, food field, environmental field, biotechnology, diagnostic reagent and protein chip, biotechnology. Identification of proteins artificially produced using technology can be used for refolding processes.

Claims

請求の範囲  The scope of the claims
[I] 標的タンパク質に可逆的に結合する認識部位を有し、  [I] has a recognition site that reversibly binds to the target protein,
上記認識部位は、上記標的タンパク質に結合可能な金属を有する金属錯体、およ び、上記標的タンパク質の基質阻害剤の少なくとも一方を有することを特徴とするィ ンプリントポリマー。  The imprinted polymer, wherein the recognition site has at least one of a metal complex having a metal capable of binding to the target protein and a substrate inhibitor of the target protein.
[2] 上記金属錯体は、標的分子と結合することによって結合シグナルを発することを特 徴とする請求項 1に記載のインプリントポリマー。  [2] The imprinted polymer according to [1], wherein the metal complex emits a binding signal by binding to a target molecule.
[3] 請求項 1または 2に記載のインプリントポリマーを備えるアレイチップ。 [3] An array chip comprising the imprinted polymer according to claim 1 or 2.
[4] 請求項 1または 2に記載のインプリントポリマーに対する吸着特性の違いからタンパ ク質を同定する方法。 [4] A method for identifying a protein from the difference in adsorption characteristics with respect to the imprinted polymer according to claim 1 or 2.
[5] 請求項 1または 2に記載のインプリントポリマーに対する吸着特性の違いからタンパ ク質を精製する方法。  [5] A method for purifying a protein from the difference in adsorption characteristics with respect to the imprinted polymer according to claim 1 or 2.
[6] 請求項 1または 2に記載のインプリントポリマーによってタンパク質をリフォールディ ングする方法。  [6] A method for refolding a protein with the imprinted polymer according to claim 1 or 2.
[7] 請求項 1または 2に記載のインプリントポリマーによってタンパク質の折りたたみ状態 を判別する方法。  [7] A method for determining a protein folding state using the imprinted polymer according to claim 1 or 2.
[8] 標的タンパク質と機能性モノマーとの複合体を形成する複合体形成工程と、  [8] a complex forming step for forming a complex of the target protein and the functional monomer;
上記複合体中の機能性モノマーを重合させる重合工程とを含み、  A polymerization step of polymerizing the functional monomer in the complex,
上記機能性モノマーは、標的タンパク質と結合可能な金属に配位する配位子、お よび、標的タンパク質の基質阻害剤の少なくとも一方を含むことを特徴とするインプリ ントポリマーの製造方法。  The method for producing an imprint polymer, wherein the functional monomer includes at least one of a ligand coordinated to a metal capable of binding to a target protein and a substrate inhibitor of the target protein.
[9] 上記複合体形成工程によって形成された複合体を単離する単離工程を含み、当 該単離工程後に重合工程を行うことを特徴とする請求項 8に記載のインプリントポリマ 一の製造方法。 [9] The imprint polymer according to claim 8, further comprising an isolation step of isolating the complex formed by the complex formation step, and performing a polymerization step after the isolation step. Production method.
[10] 上記複合体形成工程および重合工程の少なくとも一方を行った後に、上記金属に 配位可能な配位子によって、タンパク質と相補的な位置力も外れた金属をブロックす ることを特徴とする請求項 8または 9に記載のインプリントポリマーの製造方法。  [10] After performing at least one of the complex formation step and the polymerization step, the ligand that can coordinate with the metal is used to block the metal that has also lost its complementary position to the protein. The method for producing an imprinted polymer according to claim 8 or 9.
[II] 上記重合工程を行った後、 キレート剤を添加すること、又は pHを変化させることによって上記配位子力 金属 を脱離させ、その後、上記金属とは異なる金属を配位させることによって、上記インプ リントポリマーの機能改変を行う工程を含むことを特徴とする請求項 8〜10のいずれ 力 1項に記載のインプリントポリマーの方法。 [II] After performing the polymerization step, A step of modifying the function of the imprint polymer by adding a chelating agent or desorbing the ligand force metal by changing the pH and then coordinating a metal different from the metal. The method of imprinting polymer according to any one of claims 8 to 10, characterized in that
[12] 標的タンパク質と当該標的タンパク質の基質とを複合化させた基質一タンパク質複 合体と、機能性モノマーとを複合化させる工程と、上記工程後に機能性モノマーを重 合させる工程を行うことを特徴とするタンパク質の固定ィ匕方法。  [12] A step of complexing a substrate-protein complex in which a target protein and a substrate of the target protein are complexed with a functional monomer, and a step of superimposing the functional monomer after the above steps are performed. Characterized protein immobilization method.
[13] 上記機能性モノマーは、上記標的タンパク質と結合可能な金属に配位する配位子 を含むことを特徴とする請求項 12に記載のタンパク質の固定ィ匕方法。  13. The protein immobilization method according to claim 12, wherein the functional monomer includes a ligand that coordinates to a metal that can bind to the target protein.
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