JP2014210733A - Protein-immobilized carrier and method for producing same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein-immobilized carrier in which efficiency of binding between a functional protein immobilized on a carrier and a substance specifically bound with the functional protein is improved.SOLUTION: There is provided the protein-immobilized carrier which has a functional protein, a peptide linker and a carrier. The functional protein has amino acid residues of the N terminal having cysteine residues, glycine residues or alanine residues and has a function of specifically binding with a specific substance. The peptide linker is formed by linking one or more kind of amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, tryptophan, methionine, glycine, threonine, histidine, and cysteine. The carrier supports the functional protein through the peptide linker. The carrier and the N terminal of the peptide linker are covalently bonded, and the C-terminal of the peptide linker and the N terminal of the functional protein are bonded by amide bond.

Description

  The present invention relates to a protein-immobilized carrier and a method for producing the same. Specifically, the present invention relates to a protein-immobilized carrier in which a functional protein having a function of specifically binding to a specific substance is immobilized on a carrier, and a method for producing the same.

  There is an immobilization method as a method for stably utilizing protein functions in vitro. The immobilization method is a technique for immobilizing and retaining the protein to be used in the reaction system, and is a carrier binding method in which the protein is bound to an insoluble carrier through a chemical bond. Examples include inclusion methods, cross-linking methods in which proteins are linked to form insoluble macromolecules. In particular, the carrier binding method has the advantages of stabilizing the immobilized protein and facilitating continuous use of the carrier. Therefore, a functional protein having a function of specifically binding to a specific substance such as an antibody or an enzyme is immobilized by a carrier binding method and applied as a stationary phase of affinity chromatography, a detector of a biosensor, or the like. Yes.

  As an example of an affinity chromatography stationary phase, Japanese Patent Application Laid-Open No. 2012-001462 (Patent Document 1) discloses an inorganic monolith in which a protein having antibody binding ability is immobilized via an amino group or a thiol group of the protein. Techniques related to this are disclosed.

JP 2012-001462 A

In Patent Document 1, as a method for immobilizing an antibody-binding protein to an inorganic monolith via an amino group or thiol group of a protein, an epoxy group is introduced into the inorganic monolith, and the amino group or thiol of the protein is compared with the epoxy group. Addition reaction of group, introduction of leaving group into inorganic monolith, nucleophilic substitution reaction with amino group or thiol group of protein, introduction of aldehyde (formyl group) into inorganic monolith, Examples include a method of performing a reductive amination reaction (reducing and forming an imine after forming an imine), and a method of introducing a thiol group into an inorganic monolith to form a disulfide bond with a protein thiol group. (Page 6, line 44 to page 7, line 7; see paragraph [0036] of the specification).
However, in such a method, a large number of proteins to be immobilized may be bonded to the carrier via different functional groups, and each protein is not necessarily uniformly oriented in a predetermined direction. In particular, when a functional protein having a function of specifically binding to a specific substance is immobilized on a carrier, the functional protein is immobilized without a binding site for the substance being presented on the surface side of the carrier. It may be done. Since the ability of such a functional protein to bind to a substance is impaired, the efficiency with which a protein-immobilized carrier binds to the substance with the generation of a functional protein that is not properly oriented is determined by the volume of the carrier. There is a problem that it falls.
Accordingly, an object of the present invention is to provide a protein-immobilized carrier in which the efficiency of binding between a functional protein immobilized on a carrier and a substance to which the functional protein specifically binds is improved.

In order to solve the above problems, the protein-immobilized carrier according to the present invention is:
A functional protein in which the N-terminal amino acid residue has a cysteine residue, a glycine residue or an alanine residue and has a function of specifically binding to a specific substance;
A peptide linker formed by linking one or more amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, tryptophan, methionine, glycine, threonine, histidine and cysteine;
A carrier carrying the functional protein via the peptide linker;
Having
The carrier and the N-terminus of the peptide linker are covalently bonded;
The C-terminal of the peptide linker and the N-terminal of the functional protein are amide-bonded.

  According to the present invention, it is possible to provide a protein-immobilized carrier in which the efficiency of binding between a functional protein immobilized on a carrier and a specific substance to which the functional protein specifically binds is improved.

It is a schematic diagram which shows an example of the structure of the protein fixed carrier which concerns on this embodiment. It is a schematic diagram which shows the binding state of the functional protein and support | carrier in the protein fixed support | carrier of a comparative example. It is a figure which shows an example of the manufacturing method of the protein fixed carrier which concerns on this embodiment. It is a figure which shows an example of the process of fixing a peptide linker to a support | carrier. It is a figure which shows an example of the process of linking a functional protein to a peptide linker. It is a figure which shows the other example of the process of connecting a functional protein to a peptide linker. It is a figure which shows the process of desulfurizing the thiol group which a functional protein has. It is a figure which shows an example of the isolation | separation purification apparatus using the protein fixed support | carrier which concerns on this embodiment as a isolation | separation purification material. It is a figure which shows the efficiency of the coupling | bonding with respect to the target substance of the protein fixed support which concerns on an Example.

  Hereinafter, the protein-immobilized carrier and the production method thereof according to the embodiment of the present invention will be described in detail.

FIG. 1 is a schematic diagram showing an example of the structure of a protein-immobilized carrier according to this embodiment.
As shown in FIG. 1, the protein-immobilized carrier 1 according to an embodiment of the present invention has a functional protein 50 having a function of specifically binding to a specific substance via a peptide linker 40. It is fixed to.
The protein-immobilized carrier 1 is formed by immobilizing a functional protein 50 having a function of specifically binding to a specific substance on the carrier 30, thereby recognizing and binding the substance as the target substance 60. It is a protein-carrier complex that enables the function to be provided stably and continuously in vitro.
In the protein-immobilized carrier 1 according to this embodiment, the functional protein 50 and the peptide linker 40 form a unit structure that is a substantially linear branch on the carrier 30. Such a unit structure is repeatedly provided on the surface of the protein-immobilized carrier 1. The functional protein 50 is located on the outermost surface side of the protein-immobilized carrier 1 and is supported on the carrier 30 so that it can easily interact with the target substance 60 in contact with the protein-immobilized carrier 1.
Specifically, such a protein-immobilized carrier 1 is a functional protein 50 in which a desired protein is immobilized, so that a specific substance to which the protein specifically binds is separated from a mixture. It can be used as a purification material. In addition, the functional protein 50 can be used as an enzyme immobilization carrier by immobilizing a desired enzyme.

FIG. 2 is a schematic diagram showing a binding state between the functional protein and the carrier in the protein-immobilized carrier 2 of the comparative example.
In FIG. 2, as an example of the functional protein 50, a plurality of antibodies (immunoglobulins) are immobilized on the carrier 30. An antibody is a protein that has a binding site on the tip end side of a branch shown in a Y shape and specifically binds an antigen as the target substance 60 to this site.
The functional protein 50 immobilized on the protein-immobilized carrier 2 of the comparative example may have a plurality of functional groups capable of binding to the carrier 30 in the molecule. Therefore, each functional protein 50 to be immobilized is bonded to the carrier 30 via an arbitrary functional group in the molecule, and each of the plurality of functional proteins 50 is in a predetermined direction as shown in FIG. It will be carried on the carrier 30 without being uniformly oriented. A part of the functional protein 50 oriented and immobilized in an arbitrary direction is buried inside without exposing the binding site of the functional protein 50 to the surface of the carrier 30 and cannot bind to the target substance 60. It becomes a state.
Therefore, in the protein-immobilized carrier 2 of the comparative example in which the orientation of the functional protein 50 is not controlled, the functional protein 50 and the target substance per the surface area of the carrier depending on the proportion of the functional protein 50 that is not properly oriented. The effective area of interaction with 60 is reduced, and the efficiency with which the protein-immobilized carrier 2 is bound to the target substance 60 is reduced when viewed per volume of the carrier.

Therefore, in the protein-immobilized carrier 1 according to this embodiment, as shown in FIG. 1, the peptide linker 40 is covalently bonded to the carrier 30 at the N-terminus, and the N-terminus of the functional protein 50 is connected to the C-terminus of the peptide linker 40. The functional protein 50 is supported by being uniformly oriented in a predetermined direction with respect to the carrier 30 by the unit structure formed by amide bond.
In the protein-immobilized carrier 1, as shown in FIG. 1, each peptide linker 40 supported on the carrier 30 has a functional group at the same position, that is, an amino group of the N-terminal main chain of the peptide linker 40. Via the carrier 30. In addition, each functional protein 50 is amide-bonded with the functional group at the same position, that is, the carboxyl group of the C-terminal main chain of the peptide linker 40 through the amino group of the N-terminal main chain of the functional protein 50. Yes.
Therefore, each unit structure formed on the carrier 30 forms a substantially linear branch having a similar molecular structure, and each of the plurality of functional proteins 50 carried at the ends of the branches is N From the terminal side toward the C terminal side, the film is uniformly oriented in a predetermined direction.
In FIG. 1, the covalent bond formed by the peptide linker 40 and the carrier 30 is an amide bond.
In addition, the N-terminal amino acid residue of the functional protein 50 involved in the binding between the functional protein 50 and the peptide linker 40 is a cysteine residue. This cysteine residue can be a glycine residue or an alanine residue depending on the production method.

  Next, the configuration of the protein-immobilized carrier 1 according to this embodiment will be described.

The carrier 30 according to the present embodiment is a solid phase carrier that carries the functional protein 50 via the peptide linker 40. The carrier 30 has a plurality of functional groups on the surface, and forms a covalent bond with the amino group of the N-terminal main chain of the peptide linker 40 via these functional groups.
Examples of the functional group that the carrier 30 has include a carboxyl group or an amino group, and a carboxyl group in which a reaction for forming a covalent bond is more common is particularly preferable.
As the material of the carrier 30, an organic polymer or an inorganic polymer that forms an insoluble polymer can be used depending on the use of the protein-immobilized carrier 1 to be produced.

Examples of organic polymer materials include polysaccharides such as agarose, cellulose, and heparin, polyolefins such as polyethylene and polypropylene, polystyrene resins such as polystyrene and polyhydroxystyrene, chlorine resins such as polyvinyl chloride and polyvinylidene chloride, Appropriate materials such as polyester such as acrylamide and polyethylene terephthalate, nylon, aramid, urea resin, melamine resin, and polycarbonate can be used. In addition, these organic polymers may be modified in the constituent monomers, and may be copolymers or graft polymers with other molecules.
Inorganic polymer materials include inorganic materials such as soda glass, quartz and silica gel, organopolysiloxanes such as polydimethylsiloxane, metals such as gold and titanium, inorganic carbon such as graphene, and other materials such as ceramics. can do.

The carrier 30 made of these polymers is prepared so that a functional group capable of forming a covalent bond with the N-terminal amino group of the peptide linker 40 is introduced on the surface of the carrier 30. For example, the organic polymer carrier 30 can be prepared by polymerizing a monomer having a predetermined functional group. In this case, the functional group may be present in at least one of the end of the main chain and the side chain in the polymer structure.
Moreover, a functional group can be introduced on the surface of the carrier 30 by chemically treating the polymer. For example, as a method for introducing a carboxyl group as a functional group, a method in which a halogenated acetate is reacted with a carrier 30 having a hydroxyl group in the presence of a high concentration alkali (see, for example, JP-A-1-262468), A method of reacting a carrier 30 having a dicarboxylic acid anhydride (for example, see US Pat. No. 4,560,704), a method of reacting a carrier 30 having an epoxy group with an amino compound having a carboxyl group such as aminocaproic acid, and an allyl group. An appropriate method such as a method of oxidizing the carrier 30 having permanganate with a permanganate (for example, see US Pat. No. 4,029,583) can be used. Alternatively, a functional group can be introduced into an organic polymer or an inorganic polymer by using a chemical treatment agent such as a silane coupling agent having a plurality of functional groups in the molecule. In this case, one of the functional groups of the chemical treatment agent may be a desired functional group capable of forming a covalent bond with the N-terminal amino group of the peptide linker 40, and the other may be a functional group capable of reacting with the carrier 30.
Further, by forming a monomolecular film of an organic polymer on the surface of the inorganic polymer, a functional group can be introduced on the surface of the carrier 30 made of the inorganic polymer. In this case, a desired functional group capable of forming a covalent bond with the N-terminal amino group of the peptide linker 40 may be introduced so as to be arranged on the opposite side of the adsorption site of the molecule forming the monomolecular film.

  The shape of the carrier 30 can be an appropriate shape according to the use of the protein-immobilized carrier 1 such as a plate shape, a sheet shape, a bead shape, a membrane shape, and a fiber shape.

The peptide linker 40 according to the present embodiment is a linker that connects between the functional protein 50 and the carrier 30, and a plurality of predetermined amino acids having no amino group or carboxyl group in the side chain are connected via an amide bond. It is a peptide formed.
The peptide linker 40 has a substantially linear structure having an N-terminal main chain amino group as a unique amino group and a C-terminal main chain carboxyl group as a unique carboxyl group. Only the amino group of the N-terminal main chain is covalently bonded to the carrier, and only the carboxyl group of the C-terminal main chain is bonded to the amino group of the N-terminal main chain of the functional protein 50, whereby a plurality of functional proteins 50 are predetermined. It is fixed to the carrier 30 so as to be uniformly oriented in this direction.

Examples of amino acids constituting the peptide linker 40 include one or more amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, tryptophan, methionine, glycine, threonine, histidine and cysteine. It is done. Since the peptide linker 40 is composed of amino acids having no amino group or carboxyl group in these side chains, the peptide linker molecule has only one amino group at the N-terminus and only one carboxyl group at the C-terminus. Will be provided.
The amino acid constituting the peptide linker 40 may be either a D-form amino acid or an L-form amino acid, and the peptide linker 40 is a polyamino acid in which only either the D-form amino acid or the L-form amino acid is linked, or the D-form amino acid. These may be any of polyamino acids in which the amino acids and L-amino acids are linked in combination.

  The amino acid sequence of the peptide linker 40 may be any sequence, and may be either a homopolymer composed of one type of amino acid or a heteropolymer composed of a combination of a plurality of types of amino acids. In addition, the three-dimensional structure formed by the peptide linker 40 may be any shape such as a linear shape, a sheet shape, a spherical shape, etc. Well, it is not limited to a structure composed of an α-helix, β-sheet or a motif formed by a combination thereof. Since the C-terminal amino acid of the peptide linker 40 affects the efficiency of linking to the functional protein 50, among the amino acids described above, amino acids other than valine, isoleucine, and threonine are preferable.

  The length of the peptide linker 40 is preferably a length in which 3 or more and 200 or less amino acid residues are linked, and is preferably 6 mm (0.6 nm) or more. More preferably, the length is 3 to 100 amino acid residues, more preferably 3 to 50 amino acid residues linked. When the length of the amino acid sequence of the peptide linker 40 is less than the length in which three amino acid residues are linked, the efficiency of binding the functional protein 50 to the carrier 30 tends to decrease. On the other hand, when the length of the amino acid sequence of the peptide linker 40 exceeds the length in which 200 amino acid residues are linked, the three-dimensional structure of the peptide linker 40 in the solution becomes unstable and is not maintained in a certain structure. Therefore, there is a possibility that the functional protein 50 cannot be oriented in a predetermined direction.

The functional protein 50 according to the present embodiment is a protein having a function of specifically binding to a specific substance.
“Specifically binds” means that binding affinity to a certain substance is recognized, but binding affinity to other substances is remarkably low, so that the substance can selectively bind only to the specific substance. To do. Bonds include both reversible and irreversible bonds. In addition to covalent bonds, hydrogen bonds, hydrophobic bonds, van der Waals forces, ionic bonds, etc. Includes the formation of action.
The specific substance that specifically binds is not limited to a single substance, but includes a group of substances having similar properties and a plurality of dissimilar substances.

  The functional protein 50 may be a natural protein or an artificial protein as long as it has a function of specifically binding to a specific substance. Such a function is not limited to the case where the functional protein 50 is provided for the original role to be played in the living body, but may be provided as one property.

  Examples of the functional protein 50 are, for example, protein A derived from the cell wall of Staphylococcus aureus, protein G derived from the cell wall of a microorganism belonging to the genus Streptococcus, and Peptostreptococcus magnus. Antibody binding proteins such as protein L, sugar binding proteins such as lectin, heparin binding protein and chitin binding protein, lipid binding proteins such as fatty acid binding protein and phospholipid binding protein, nucleic acid binding proteins such as DNA binding protein and RNA binding protein, Inorganic binding protein such as metal binding protein and inorganic ion binding protein, protein binding protein, glycoprotein binding protein, lipoprotein binding protein, amino acid binding protein, Vita Down binding proteins, antigens, avidin, streptavidin, other enzymes or receptor proteins, and the like.

The functional protein 50 according to this embodiment is amide-bonded to the carboxyl group of the C-terminal main chain of the peptide linker 40 via the amino group of the N-terminal main chain. When the functional protein 50 is connected to the peptide linker 40, the N-terminal amino acid residue is composed of cysteine, glycine or alanine. As described above, the reason that the N-terminal amino acid of the functional protein 50 is limited to a predetermined amino acid is that it is allowed to link the functional protein 50 and the peptide linker 40 in the method for producing the protein-immobilized carrier 1 described later. This is because N-terminal amino acids are limited.
In the protein-immobilized carrier 1 according to the present embodiment in which the functional protein 50 is bound to the carrier 30 via the peptide linker 40, N of one molecule of the functional protein 50 is attached to the C terminal of the peptide linker 40 of one molecule. The chain peptide formed by bonding the ends has a structure in which the carboxyl group or amino group of the carrier 30 is uniformly bonded to preferably 50% or more, more preferably 70% or more. In addition, a large number of such chain-like peptides to be supported are uniformly 70% or more, more preferably 80% or more, and still more preferably 90% or more via functional groups each having the same site. Are connected to each other.

In the protein-immobilized carrier 1 according to this embodiment having such a structure, the plurality of functional proteins 50 immobilized on the carrier 30 are uniformly oriented in a predetermined direction. Therefore, the binding site of the functional protein 50 is appropriately embedded on the surface side of the carrier 30 without being buried inside the carrier 30. Therefore, the ratio of the functional protein 50 supported in a state capable of interacting with the target substance 60 in the protein-immobilized carrier 1 is improved, and the efficiency of specifically binding the protein-immobilized carrier 1 to the target substance 60 is inappropriate. Compared to the carrier in which the functional protein 50 is immobilized in a simple orientation, this is improved.
Further, in the protein-immobilized carrier 1 having such a structure, each functional protein 50 is regularly arranged and immobilized on the carrier 30, so that it is less likely to cause steric hindrance and is supported adjacently. Interference with other functional protein 50 is reduced. Furthermore, the functional protein 50 can adopt a three-dimensional structure appropriately folded like a natural protein by being immobilized on the carrier 30 on the N-terminal side.
And according to such protein immobilization support 1 concerning this embodiment, by carrying a desired protein as functional protein 50, the binding efficiency with the substance which the protein binds specifically improves, and binding Separation and purification material having an improved capacity can be provided. In addition, by supporting a desired enzyme as the functional protein 50, the efficiency with which the enzyme interacts with the substrate is improved, and an enzyme-immobilized carrier with improved enzyme reaction efficiency can be provided.

  Next, a method for producing a protein-immobilized carrier that is one embodiment of the present invention will be described.

FIG. 3 is a diagram showing an example of a method for producing a protein-immobilized carrier according to the present embodiment.
In the method for producing a protein-immobilized carrier according to the present embodiment, the N-terminal amino acid residue mainly comprises an amino acid or cysteine to which a detachable modifying group containing a thiol group is bonded, and a specific A step of preparing a functional protein having a function of specifically binding to a substance, and selected from the group consisting of alanine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, tryptophan, methionine, glycine, threonine, histidine and cysteine A step of preparing a peptide linker in which one or a plurality of amino acids are linked and the C-terminal carboxyl group is thioesterified (hereinafter, the step of preparing these functional protein and peptide linker is referred to as the preparation step) And have a plurality of carboxyl groups on the surface. The body and the N-terminus of the prepared peptide linker are fixed (hereinafter also referred to as the fixing step), and the C-terminus of the prepared peptide linker and the N-terminus of the functional protein are bonded with an amide bond. And a step of connecting (hereinafter also referred to as a connecting step).
FIG. 3 shows a method of immobilizing the peptide linker 40 on the carrier 30 and then linking the functional protein 50 to the peptide linker 40 immobilized on the carrier 30. A method for producing this protein-immobilized carrier is shown in FIG. In each of the fixing step and the connecting step, the order of the execution order is not limited. That is, instead of the procedure of FIG. 3, a method in which the functional protein 50 is linked to the peptide linker 40 and then the peptide linker 40 linked to the functional protein 50 is immobilized on the carrier 30 may be employed. However, when the functional protein 50 has a functional group capable of binding to the carrier 30 in the middle part of the amino acid sequence excluding the N-terminus or C-terminus, the linking step may be performed after the fixing step. preferable.
In FIG. 3, the covalent bond formed by the peptide linker 40 and the carrier 30 is an amide bond, and the N-terminal amino acid residue of the functional protein 50 involved in the binding between the functional protein 50 and the peptide linker 40 is left. The group is a cysteine residue, but is not limited thereto.

In the preparation step, a peptide linker in which predetermined amino acids are linked and the C-terminal carboxyl group is thioesterified is prepared (FIG. 3 (a1)).
The peptide linker 40 in which the C-terminal carboxyl group is thioesterified can be prepared by synthesizing a peptide using a known chemical synthesis method and then thioesterifying, but can directly obtain a peptide thioester. It is preferable to use either a method using splicing or a peptide synthesis method using a solid phase with a predetermined modification.

  A method using intein splicing is a method using intesplicing induced by a thiol compound after expressing a peptide as an intein-binding peptide by using genetic engineering techniques (Chong, S. et al., Gene 1997, 192, 2, 271 etc.). When the peptide linker 40 is produced as a fusion peptide in which intein is bound to the C-terminal in a predetermined host and then self-splicing is induced by induction of a thiol compound, the peptide linker 40 released in a state where the C-terminal carboxyl group is thioesterified. Is obtained. Specifically, an IMPACT (registered trademark) Kit (manufactured by New England BioLabs) or the like in which a Bacillus circulans chitin-binding domain (CBD) is bound to an intein of Saccharomyces cerevisiae can be used.

  The peptide synthesis method using a solid phase with a predetermined modification is a method of performing solid phase peptide synthesis using a resin modified with a predetermined thio compound as a solid phase. In this method, first, the C-terminal amino acid to be thioesterified is bound to a resin having a predetermined modification, and then the peptide is extended in the direction from the C-terminal to the N-terminal according to a conventional method. After the synthesis of the peptide having the desired sequence, when the synthesized peptide is cleaved from the resin, the peptide linker 40 released in a state in which the C-terminal carboxyl group is thioesterified is obtained. As such a method, specifically, a method of performing solid phase peptide synthesis by Boc method using a thioester resin as a solid phase (see Dawson, PE et al., Science, 1994, 266, 766, etc.), acylsulfonamide resin The solid phase peptide synthesis by Fmoc method (see Ingenito, R. et al., J. Am. Chem. Soc., 1999, 121, 49, 11369, etc.) is used.

  Also, instead of directly preparing a peptide linker in which the C-terminal carboxyl group is thioesterified, the thioacid of the peptide linker 40 is prepared, and the thioacid is reacted with an alkyl halide to react with the C-terminal carboxyl group. A thioesterified peptide linker 40 can also be obtained. For example, as a method for preparing a peptide thioic acid, a solid phase peptide synthesis method using a resin in which a dicyclohexylamine salt is immobilized by reacting an N-hydroxysuccinimide amino acid ester with benzylthiol (Lynne, EC et al. , Tetrahedron lett, 1995, 36, 8, 1217 etc.). When the synthesized peptide is separated from the carrier with hydrogen fluoride, the thioacid of the peptide linker 40 is obtained, and when this thioacid is reacted with benzyl bromide or the like, a peptide thioester can be obtained.

  The thioesterified peptide linker 40 to be prepared includes a benzyl group, 4-methylbenzyl group, 4-methoxybenzyl group, 2,4,6-trimethoxybenzyl group, diphenylmethyl group, trityl group, tert-butyl group, Peptide linkers that are thioesterified with a tert-butylsulfanyl group, 9-fluorenylmethyl group or the like as a substituent can be mentioned, and a benzylthioester is preferred.

  The preparation step further includes a functional protein 50 having a function of specifically binding to a specific substance, wherein the N-terminal amino acid residue is formed from an amino acid to which a detachable modifying group including a thiol group is bound. Or a functional protein 50 in which the N-terminal amino acid residue is cysteine (FIG. 3 (a2)). As the functional protein 50, a desired functional protein 50 is selected according to the use of the protein-immobilized substrate 1 to be manufactured, and the preparation varies depending on whether or not the N-terminal amino acid of the functional protein 50 is cysteine. The method is used.

The functional protein 50 in which the N-terminal amino acid is cysteine in the natural amino acid sequence is obtained by subjecting a protein having the same sequence as the natural amino acid sequence to a chemical synthesis method or a biochemical synthesis method without modifying the N-terminal amino acid. And can be prepared by synthesis.
As the chemical synthesis method, a known method such as a solid phase peptide synthesis method in which a protected amino acid is reacted with an amino acid on a solid phase to extend a polyamino acid chain by chemical synthesis can be used.
In addition, as a biochemical synthesis method, a method of translating a gene using the action of a protein synthase can be used. For example, the functional protein 50 is obtained by introducing and expressing a gene of the functional protein 50 in a predetermined host cell, producing the functional protein 50 in the host cell, and then separating and purifying from the host cell.
When the functional protein 50 is a naturally occurring protein, it can be prepared by separating and purifying the naturally occurring protein from the source organism. In this case, the functional protein 50 can be obtained from the source organism that produces the functional protein 50 by separation and purification according to a conventional method. In addition, the functional protein 50 can be obtained by synthesizing a protein having the same sequence as the natural amino acid sequence using a known chemical synthesis method.

On the other hand, the functional protein 50 in which the N-terminal amino acid is not cysteine in the natural amino acid sequence can be prepared by modifying so that the N-terminal amino acid is cysteine.
As a method of changing the N-terminal amino acid to cysteine, the method of substituting the amino acid at the N-terminal of the functional protein 50 with cysteine, the method of deleting the amino acid present on the N-terminal side from the first cysteine appearing in the amino acid sequence, Alternatively, a method of adding cysteine to the N-terminus of the amino acid sequence or a method of adding a polyamino acid having a cysteine at the N-terminus can be used as long as the function of the functional protein 50 is not impaired.
An appropriate method can be used for such an amino acid modification. For example, a method of introducing a site-specific mutation into DNA encoding the functional protein 50 using a genetic engineering technique can be used. . Site-directed mutagenesis methods include the Kunkel method (PNAS, 1985, 82, 488), inverse PCR method, and other methods described in “Molecular Cloning (4th Edition)” (Cold Spring Harbor Laboratory Press, 2012 Chapter 14). Etc. can be used.
The DNA encoding the functional protein 50 introduced with site-specific mutation is introduced into a predetermined host cell and expressed, and after the functional protein is produced in the host cell, the N-terminal amino acid is separated and purified. Obtains functional protein 50 modified to cysteine.

In addition, for functional proteins in which the N-terminal amino acid is not cysteine in the natural amino acid sequence, a chemical synthesis method is used so that the N-terminal amino acid residue is an amino acid to which a detachable modifying group containing a thiol group is bound. Can be prepared.
As such a method, an N- (2-mercaptoethoxy) group is introduced into the N-terminus of a functional protein as a leaving modifying group containing a thiol group (Canne, L. et al., J. Am Chem. Soc., 1996, 118, 5891) can be used. The N-terminal amino acid of the functional protein into which a detachable modifying group containing a thiol group is introduced can be glycine.

FIG. 4 is a diagram showing an example of a process for fixing a peptide linker to a carrier.
In the fixing step, a covalent bond is formed between the carboxyl group or amino group of the carrier and the N-terminal amino group of the peptide linker.
In order to orient the functional protein 50 in a predetermined direction in the protein-immobilized carrier 1 to be produced, in order to avoid denaturation or thermal decomposition of the carrier or peptide linker, a covalent bond may be formed under mild conditions. preferable. Therefore, as a method of forming a covalent bond between the carboxyl group of the carrier 30 and the peptide linker 40, a condensation reaction using a condensing agent such as a phosphonium compound, a uronium compound, an imonium compound, or a carboximide compound is used. As a method for forming a covalent bond between the amino group having the amino group and the peptide linker 40, a crosslinking reaction with glutaraldehyde is used.

  Examples of phosphonium compounds include benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), and bromo-trispyrrolidino. Phosphonium hexafluorophosphate (PyBroP), (7-azabenzotriazol-1-yl) -oxytrisdimethylaminophosphonium hexafluorophosphate (AOP), (7-azabenzotriazol-1-yl) -oxytris (pyrrolidino) Examples thereof include phosphonium hexafluorophosphate (PyAOP).

  Examples of uronium compounds include O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU), O- (benzotriazol-1-yl) -N. , N, N ′, N′-tetramethyluronium tetrafluoroborate (TBTU), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluoro Phosphate (HATU), O- (benzotriazol-1-yl) -N, N, N ′, N′-bis (tetramethylene) uronium hexafluorophosphate (HBPyU), O- (benzotriazole-1- Yl) -oxybis (pyrrolidino) uronium hexafluorophosphate (HAPyU) and the like.

  Examples of imonium compounds include benzotriazol-1-yloxy-N, N-dimethylmethaneiminium hexachloroantimonate (BDMP) and 1- (1H-benzotriazol-1-yloxy-phenylmethylenepyrrolidinium hexachloroantimonate (BPMP). Etc.

  As the carbodiimide compound, N, N-dicyclohexylcarbodiimide (DCC), N, N-diisopropylcarbodiimide (DIC), phenylethylcarbodiimide (PEC), phenylisopropylcarbodiimide (PIC), N-tert-butyl-N′-methylcarbodiimide (BMC), N-tert-butyl-N′-ethylcarbodiimide (BEC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), N, N′-di-tert-butylcarbodiimide, N, N′-di-p-tolylcarbodiimide, N-cyclohexyl-N ′-(2-morpholinoethyl) carbodiimide, and the like.

  Among these condensing agents, in the condensation reaction using the most frequently used carbodiimide compound, a urea compound is produced as a by-product, so that such a by-product is separated and removed from the condensation reaction product obtained after the reaction. Need to do. Therefore, as shown in FIG. 4, by using a water-soluble carbodiimide compound (Water Soluble Carbodiimide; WSC) as a condensing agent, a by-product can be generated as a soluble compound. This soluble by-product can be easily separated and removed by washing.

The condensation reaction for fixing the peptide linker 40 to the carrier 30 using a water-soluble carbodiimide compound is performed as follows.
First, the water-soluble carbodiimide compound 70 is added to the carrier 30 having a carboxyl group (FIG. 4A).
Then, the water-soluble carbodiimide compound 70 is added to the carboxyl group of the carrier 30 to generate an O-acylated carbamide derivative, and the carrier 30 is activated (FIG. 4B). At this time, a part of the water-soluble carbodiimide compound 70 produces an N-acylated carbamide derivative as a by-product, and the remainder remains unreacted. Therefore, by washing the carrier 30, the by-product and the unreacted water-soluble carbodiimide compound 70 are removed.
Next, the peptide linker 40 is added to the activated carrier 30 (FIG. 4C). The activated O-acylated carbamide derivative on the carrier 30 forms an electrophilic acid anhydride between the carboxyl groups on the carrier 30, and the nucleophilic N included in the peptide linker 40 is provided on this acid anhydride. When the terminal amino group is added, the peptide linker 40 is fixed to the carrier 30 (FIG. 4D).
The carrier 30 on which the peptide linker 40 is immobilized is washed to remove the unreacted peptide linker 40 and is used for the subsequent steps.

Such a condensation reaction can be performed, for example, in the range of 4 ° C. to 40 ° C. and pH 4.0 to 7.0. A preferred temperature range is 15 ° C to 40 ° C. By performing the reaction at a temperature near room temperature, the reaction time can be shortened, and denaturation and thermal decomposition of the carrier 30 and the peptide linker 40 can be avoided. Moreover, the range of preferable pH is pH 6.0-7.0. By carrying out the reaction at a pH near neutrality, denaturation of the carrier 30 and the peptide linker 40 can be avoided.
The reaction solvent for the condensation reaction can be an aqueous phase, but it is preferable to use a non-organic acid buffer solution that maintains a preferable pH range, such as a phosphate buffer solution.
Additives can be added to the condensation reaction in order to suppress the formation of by-products and racemization. Examples of the additive include 1-hydroxy-1H-benzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).

FIG. 5 is a diagram showing an example of a step of linking a functional protein to a peptide linker.
In the linking step, an amide bond is formed between the C-terminus of the peptide linker and the amino group at the N-terminus of the functional protein.
As a method of forming an amide bond between the functional protein 50 and the peptide linker 40 in the linking step, a native chemical ligation method (see Science, 1994, 266, 776) is used.

  In an example of the linking step, the functional protein 50 in which the N-terminal amino acid residue is cysteine is chemoselective via the N-terminus of the functional protein 50 to the C-terminus of the peptide linker 40 having a thioesterified carboxyl group. Connected to

The ligation reaction for binding the functional protein 50 to the peptide linker 40 using the native chemical ligation method proceeds as follows.
First, the peptide linker 40 having a thioesterified carboxyl group and the functional protein 50 whose N-terminal amino acid is cysteine are mixed (FIG. 5A).
Then, the peptide linker 40 and the functional protein 50 undergo a thioester exchange reaction, thereby generating a reaction intermediate in which the peptide linker 40 and the functional protein 50 are linked via a thioester bond (FIG. 5B).
In the molecule of this reaction intermediate, the acyl group quickly undergoes spontaneous rearrangement (S → N rearrangement), whereby the peptide linker 40 and the functional protein 50 are linked via an amide bond (FIG. 5C). ).
Thus, the amide bond formed between the peptide linker 40 and the functional protein 50 is linked by linking the peptide linker 40 and the functional protein 50 whose N-terminal amino acid is cysteine using the native chemical ligation method. The structure can be the same as that of the amide bond between Xaa and Cys in the natural peptide, and the introduction of a non-natural modification group or the like into the amino acid residue corresponding to the N-terminus of the functional protein 50 can be avoided. .

FIG. 6 is a diagram showing another example of the step of linking a functional protein to a peptide linker.
In another example of the linking step, the functional protein 50 in which the N-terminal amino acid residue is an amino acid to which a detachable modifying group containing a thiol group is bonded has a thioesterified carboxyl group. It is chemoselectively linked to the C-terminus via the N-terminus of functional protein 50. In FIG. 6, an N- (2-mercaptoethoxy) group is bonded to the N-terminal amino acid of the functional protein 50 as a leaving modifying group containing a thiol group.

The ligation reaction in this example proceeds in the same manner as the reaction for linking the functional protein 50 whose N-terminal amino acid is cysteine (FIGS. 6A and 6B).
After the transesterification reaction, the product formed by spontaneous rearrangement of the acyl group has a structure linked via a modified amino acid having an N- (2-mercaptoethoxy) group (FIG. 6 (c)).

These ligation reactions can be performed, for example, in an aqueous phase in the range of 4 ° C to 40 ° C.
The pH in the ligation reaction is usually pH 6.0 to 8.0, preferably pH 7.0 to 7.5. If the pH is below 6.0, the reaction rate may be greatly reduced. On the other hand, when pH exceeds 8.0, there exists a possibility that a peptide and protein may carry out alkali denaturation.
In the ligation reaction, a thiol compound that promotes a thioester exchange reaction can be used as a catalyst. Examples of the thiol compound to be used include thiophenol, benzyl mercaptan, 4-mercaptophenylacetic acid, 2-mercaptoethanesulfonate and the like.
The reaction solvent for the ligation reaction is preferably a reducing atmosphere in order to avoid the formation of unintended disulfide bonds and the generation of unintended acylated compounds. Therefore, these thiol compounds can be added to the reaction solvent as a reducing agent, and tris (2-carboxyethyl) phosphine or the like can be used in combination with these thiol compounds.
Moreover, it is preferable that the reaction solvent for the ligation reaction is denatured so that the peptide or protein to be reacted is solubilized. For example, peptide or protein aggregation can be avoided by adding guanidine, which is about 6 to 7 M, to the reaction solvent.

According to the method for producing the protein-immobilized carrier 1 according to the embodiment including the above steps, each peptide linker 40 supported on the carrier 30 is connected to the carrier 30 only through the amino group of the N-terminal main chain. The protein immobilization carrier according to the embodiment in which each functional protein 50 is covalently bonded and has a structure in which only the carboxyl group of the C-terminal main chain of the peptide linker 40 is amide-bonded via the amino group of the N-terminal main chain. 1 can be manufactured.
And the some functional protein 50 carry | supported by the protein fixed support | carrier 1 which has such a structure will be in the state uniformly orientated to each predetermined direction.
Therefore, the protein-immobilized carrier 1 with improved efficiency of binding between the functional protein 50 immobilized on the carrier 30 and a substance to which the functional protein 50 specifically binds can be produced.
Further, in the fixing step, the covalent bond between the carrier 30 and the peptide linker 40 is formed by a carbodiimide compound, so that the carrier 30 and the peptide linker 40 can be prevented from being denatured in the reaction for forming the covalent bond.
Further, in the linking step, by using a native chemical ligation method, even when the functional protein 50 has other functional groups in the molecule, the amino group or carboxyl group of the functional protein 50 can be protected. Without deprotection, the peptide linker 40 and the functional protein 50 can be chemoselectively linked via only the C-terminus of the peptide linker 40 and the N-terminus of the functional protein 50. Furthermore, since racemization of amino acids can be suppressed, the orientation and the three-dimensional structure can be aligned for each of the plurality of functional proteins 50 immobilized on the carrier 30.
And in the protein fixed support | carrier 1 manufactured, it can prevent that the efficiency couple | bonded with a substance falls.

FIG. 7 is a diagram showing a step of desulfurizing a thiol group possessed by a functional protein.
In the above-described process, the functional protein 50 is linked to the peptide linker 40 using the native chemical ligation method, so that an N-terminal amino acid residue is bonded to a detachable modifying group containing a cysteine or thiol group. It is prepared to be an amino acid. Therefore, after passing through the linking step, it can be subjected to a desulfurization step of desulfurizing the thiol group of the N-terminal amino acid residue of the functional protein 50.

  When the N-terminal amino acid residue is cysteine, it can be converted to alanine by reductively desulfurizing cysteine (FIG. 7 (a)). Such desulfurization can be performed by hydrodesulfurization reaction using a desulfurization catalyst. Examples of the desulfurization catalyst include a catalyst in which a metal such as cobalt, molybdenum, nickel, tungsten, palladium, platinum, osmium, ruthenium, chromium, or an oxide thereof is supported on a porous carrier such as alumina or zeolite. . When the functional protein 50 has another thiol group in the molecule, the N-terminal cysteine may be selectively desulfurized by pre-protecting such a group.

  On the other hand, when the N-terminal amino acid residue is an amino acid residue having a detachable modifying group containing a thiol group, an unmodified amino acid is obtained by cleaving the detachable modifying group containing a thiol group. (FIG. 7B). Such cleavage can be performed by reacting the linked functional protein 50 in an acidic solvent containing zinc powder.

  According to the method for producing the protein-immobilized carrier 1 including the above desulfurization step, the produced protein-immobilized carrier 1 is produced even when the N-terminal amino acid of the functional protein 50 is not cysteine in the natural amino acid sequence. The amino acid sequence of the functional protein 50 in can be the same sequence as the natural amino acid sequence.

FIG. 8 is a diagram showing an example of a separation and purification apparatus using the protein-immobilized carrier according to the present embodiment as a separation and purification material.
The protein immobilization carrier 1 according to the present embodiment can be used as a separation and purification material for separating or purifying a specific substance recognized by the immobilized functional protein 50 from a mixture containing the substance.
This separation and purification apparatus 3 is arranged to constitute a part of the purification means in the antibody drug manufacturing apparatus that produces antibodies using animal cells. As shown in FIG. 8, the separation and purification apparatus 3 is supplied with a purified stock solution containing the antibody to be separated and purified, and the antibody is purified in the affinity column 12 packed with the protein-immobilized carrier 1 as a separation and purification material. The purified antibody is subjected to other steps as a post-purification solution.

A preceding stage of such a separation and purification apparatus 3 is provided with a culture tank for culturing animal cells that produce a specific antibody, not shown. The culture tank is provided with temperature adjusting means, pH adjusting means, aeration means for adjusting dissolved oxygen concentration and dissolved carbon dioxide concentration, and an exhaust means so that predetermined culture conditions can be maintained. Homogenized by stirring means.
In such a culture tank, when a genetically modified animal cell introduced with a specific antibody gene or an animal cell infected with a specific virus is cultured, an antibody is produced. Then, the produced culture solution containing the antibody is supplied to the separation tank, and impurities such as animal cells and viruses are removed by centrifugation, and then supplied to the culture solution receiving tank 11.

The separation and purification device 3 is a means for separating and purifying the antibody from the antibody stock solution by affinity chromatography. In general, a plurality of separation and purification apparatuses based on different principles such as an affinity chromatography apparatus, an ion exchange chromatography apparatus, and a hydrophobic chromatography apparatus are used in combination in an antibody drug production apparatus. Generally, among a plurality of separation and purification apparatuses provided in an antibody drug production apparatus, an apparatus to which an antibody stock solution is first supplied is an affinity chromatography apparatus.
As shown in FIG. 8, this apparatus includes an affinity column 12, an eluent tank 13, an equilibration liquid tank 14, and a fraction tank 15.

The affinity column 12 is a cylindrical container having openings at the upper end and the lower end, and the inside of the container is filled with the protein-immobilized carrier 1 according to the present embodiment. A functional protein 50 that specifically binds to an antibody is immobilized on the protein immobilization carrier 1.
Examples of such functional protein 50 include protein A, protein G, and protein L.

In the affinity chromatography apparatus, the affinity column is equilibrated by passing the solution from the equilibration liquid tank 14 prior to the step of separating and purifying the antibody from the antibody stock solution. The equilibration liquid tank 14 is a tank that stores a neutral buffer for equilibrating the affinity column 12.
The neutral buffer stored in the equilibration liquid tank 14 is supplied to the affinity column 12 through a pipe, and is passed through from the upper end side of the affinity column 12 so that the protein immobilized in the affinity column 12 is fixed. Protein A immobilized on the immobilized carrier 1 is equilibrated. Thereafter, the neutral buffer that has been passed through the affinity column 12 and has completed the equilibration of protein A is discharged from the lower end of the affinity column 12 and discarded through the drain 16.

In the step of separating and purifying the antibody from the antibody stock solution, the antibody stock solution is passed through the equilibrated affinity column 12 so that the antibody is roughly purified.
When the antibody stock solution temporarily stored in the culture solution receiving tank 11 is passed through the affinity column 12 from the upper end side, the antibody contained in the antibody stock solution is separated from the protein-immobilized carrier packed in the affinity column 12. The antibody is separated from the antibody stock solution containing other contaminants by contacting and specifically binding to protein A immobilized on the protein-immobilized carrier.
Thereafter, the antibody stock solution from which the antibody has been separated is discharged from the lower end of the affinity column 12 and discarded via the drain 16.

  Subsequently, the neutral buffer stored in the equilibration liquid tank 14 is supplied again to the affinity column 12 through the pipe, and is passed through from the upper end side of the affinity column 12 so that the non-binding substance is eluted. And the affinity column 12 is washed. The neutral buffer solution passed through the affinity column is discharged from the lower end of the affinity column and discarded through the drain 16.

Next, the antibody is eluted by passing the solution from the eluent tank 13. The eluent tank 13 is a tank that stores an acidic buffer for eluting the antibody bound to the affinity column 12.
The acidic buffer stored in the eluent tank 13 is supplied to the affinity column 12 through a pipe, and is passed through from the upper end side of the affinity column 12 so that the antibody that specifically binds to protein A is removed. Elute. Thereafter, the acidic buffer containing the eluted antibody is discharged from the lower end of the affinity column 12 and supplied to the fraction tank 15.

The fraction tank 15 is a tank for temporarily storing an eluate containing the antibody eluted from the affinity column 12.
In the fraction tank 15, the eluate containing the antibody is neutralized, and the purification process by the affinity chromatography apparatus is completed.

  As described above, impurities such as animal cells and viruses may remain in the eluate roughly purified in the purification step first provided after the culture. Therefore, the neutralized eluate is subjected to filtration by the filter 17 and then stored in the eluate receiving tank 18. Thereafter, it is subjected to separation and purification by another separation and purification apparatus (not shown) and further purified. The solution containing the purified antibody is concentrated in a filtration device after removing impurities such as viruses and contaminants in the purification process, and is formulated by adding a predetermined pharmaceutical aid.

In the separation and purification device 3 using the protein-immobilized carrier 1 according to this embodiment as a separation and purification material, the functional protein 50 immobilized on the protein-immobilized carrier 1 packed in the affinity column 12 is an amino acid. By being immobilized on the carrier 30 at the N-terminal side of the sequence, each of the functional proteins 50 is oriented in a predetermined direction, and the action site of the functional protein 50 is uniformly presented on the surface side of the carrier. can do.
Therefore, among the plurality of functional proteins 50 carried on the carrier 30, the ratio of the functional proteins 50 in which the function site is buried inside the carrier 30 and the function of interacting with a specific substance is reduced.
Therefore, the dynamic and static binding capacity of the protein-immobilized support 1 packed in the affinity column 12 is improved, and a separation / purification material and a separation / purification apparatus excellent in separation efficiency and purification efficiency are provided.

The protein-immobilized carrier 1 according to this embodiment can be used as an immobilized enzyme carrier by immobilizing an enzyme as the functional protein 50.
In such an immobilized enzyme carrier using the protein-immobilized carrier 1 of this embodiment, the enzyme immobilized on the carrier 30 is immobilized on the carrier 30 on the N-terminal side of the amino acid sequence, thereby Since each of these is oriented in a predetermined direction to form a protein three-dimensional structure, the enzyme action site can be uniformly presented on the surface side of the carrier 30.
Therefore, among the plurality of enzymes supported on the carrier 30, the ratio of the enzyme whose functional site is buried inside the carrier and whose function of interacting with a specific substance is reduced is reduced. Enzyme reaction efficiency is improved.

  Next, examples of the present invention will be described in detail, but the present invention is not limited thereto.

In this example, according to the method for producing a protein-immobilized carrier according to the embodiment, the protein-immobilized carrier 1 is produced, and the functional protein 50 immobilized on the carrier 30 and a substance to which the functional protein 50 specifically binds. It was confirmed whether or not the efficiency of the coupling with was improved.
A 96-well plate (manufactured by Sumitomo Bakelite Co., Ltd.) having a plurality of amino groups on the surface was used as the carrier 30, and protein A was used as the functional protein 50.
In addition, as a method for producing a protein-immobilized carrier, a method of performing a linking step after an immobilization step was adopted.

First, a peptide linker 40 in which the C-terminal carboxyl group was thioesterified was prepared. As a preparation method, a method using intein splicing was used.
First, DNA encoding the peptide linker 40 was inserted into the multi-cloning site of the vector pTXB1 of IMPACT (registered trademark) Kit (manufactured by New England BioLabs), and then this pTXB1 was transformed into E. coli ER2566 strain. This vector is an expression vector that produces a fusion protein in which the C-terminus of the target protein is bound to intein and chitin binding domain (CBD).
E. coli ER2566 strain into which pTXB1 was introduced was cultured under predetermined conditions to produce a fusion peptide, and then the obtained fusion peptide was bound to chitin beads. Subsequently, a thiol compound (dithiothreitol or 2-mercaptoethanesulfonic acid) was added, and the peptide linker 40 bound to the chitin beads was dissociated by splicing to obtain a peptide linker 40 in which the C-terminal was thioesterified.

Next, a functional protein 50 in which the N-terminal amino acid residue was cysteine was prepared. Protein A used as the functional protein 50 is a protein whose N-terminal amino acid is not cysteine. Therefore, as a preparation method, a method of adding a polyamino acid whose N-terminal is cysteine was used.
DNA encoding protein A was inserted into the multicloning site of the vector pTYB21 of IMPACT (registered trademark) Kit (manufactured by New England BioLabs), and this pTYB21 was then transformed into Escherichia coli ER2566. This vector is an expression vector for producing a fusion protein in which the N-terminus of the target protein is bound to intein and chitin binding domain (CBD).
Escherichia coli ER2566 strain into which pTYB21 was introduced was cultured under predetermined conditions to produce a fusion protein, and then the obtained fusion protein was bound to chitin beads. Subsequently, a thiol compound (dithiothreitol or 2-mercaptoethanesulfonic acid) was added and dissociated by splicing functional protein 50 bound to chitin beads to obtain functional protein 50 having an N-terminal amino acid residue consisting of cysteine. .

Next, the carrier 30 having a plurality of amino groups on the surface was bonded to the N-terminus of the prepared peptide linker 40.
First, glutaraldehyde was added to the carbonate buffer so that the final concentration was 2 v / v% to prepare a fixing solution. Next, 150 μL of the obtained fixing solution was added to each well of a 96-well plate (manufactured by Sumitomo Bakelite Co., Ltd.), and reacted at 37 ° C. for 2 hours.
After the reaction, each well was washed three times with pure water, and then 100 μL of the prepared peptide linker 40 was added to each well and reacted at 37 ° C. for 2 hours.
Thereafter, each well was washed three times with a phosphate buffer (PBS) to obtain a peptide linker 40 fixed to the carrier 30.

Next, an amide bond was made between the C-terminus of the prepared peptide linker 40 and the N-terminus of the functional protein 50.
First, a ligation reaction buffer was prepared according to the following procedure.
After dissolving 1.42 g of sodium hydrogen phosphate in deionized water, 28.7 g of guanidine hydrochloride is dissolved, so that the final concentration of sodium hydrogen phosphate is 0.1M and the final concentration of guanidine is 6M, pH 6. A 50 mL stock solution of 8-7.0 was prepared.
Then, 42.1 g of 4-mercaptophenylacetic acid (MPAA) (Sigma-Aldrich; Cat. #: 653152) and tris (2-carboxyethyl) phosphine hydrochloride (TCEP · HCl) (Sigma-Aldrich) were added to the vial. (Cat. #: C4706) 28.7 g was added, and 5 mL of the prepared stock solution was added to prepare a solution having a final concentration of MPAA of 50 mM and a final concentration of TCEP of 20 mM. At this time, the stock solution was added while filtering using a syringe filter having a pore size of 0.2 μm.
The solution to which MPAA and TCEP were added was dissolved in MPAA by adjusting the pH to around 7.0 using 1M hydrochloric acid and 2M sodium hydroxide, and then adjusted to pH 7.1 and linked. The reaction buffer was used.

Next, native chemical ligation was performed using the prepared ligation buffer.
A ligation buffer in which a functional protein 50 having an N-terminal cysteine was dissolved was added to a 96-well plate to which the peptide linker 40 having a thioesterified C-terminal was fixed, to initiate a ligation reaction. At this time, since the thioester of the peptide linker may be hydrolyzed when the pH of the ligation buffer exceeds 7, the pH of this solution was adjusted to 7.0 using 0.2 M sodium hydroxide.
Immediately after 8 hours, a phosphate buffer solution containing 1M guanidine hydrochloride aqueous solution (0.1% TFA) was immediately added to dilute the reaction solution, and the reaction solution was reduced by reducing the pH to 4 or less. The reaction was stopped.
After the reaction, each well was washed three times with a phosphate buffer (PBS) to obtain a protein-immobilized carrier 1 according to the example.

In addition, as a comparative example, a protein-immobilized carrier was produced using a method in which the functional protein 50 was directly reacted with the functional group of the carrier 30 for immobilization.
As the carrier 30 in the comparative example, a 96-well plate (manufactured by Sumitomo Bakelite Co., Ltd.) having a plurality of amino groups on the surface was used as in the example, and protein A was used as the functional protein 50.
First, glutaraldehyde was added to the carbonate buffer so that the final concentration was 2 v / v% to prepare a fixing solution. Next, 150 μL of the obtained fixing solution was added to each well of a 96-well plate (manufactured by Sumitomo Bakelite Co., Ltd.), and reacted at 37 ° C. for 2 hours.
After the reaction, each well was washed three times with pure water, and then 100 μL of functional protein 50 was added to each well and reacted at 37 ° C. for 2 hours.
Thereafter, each well was washed three times with a phosphate buffer (PBS) to obtain a protein-immobilized carrier according to a comparative example.

Next, regarding the protein-immobilized carrier 1 according to the example obtained in this way and the protein-immobilized carrier according to the comparative example, the functional protein 50 immobilized on the carrier and the functional protein 50 are specifically bound. The efficiency of interaction with certain substances was evaluated.
As a target substance 60 that specifically binds to protein A which is functional protein 50, biotin attached to AssayMax Human Tissue-Type Plasminogen Activator ELISA Kit (Cat. No. ET1001-1; manufactured by Funakoshi Co., Ltd.) Anti-human tissue plasminogen activator (t-PA) antibody (Immunoglobulin G; IgG) was used.

The measurement of the binding efficiency in the protein-immobilized carrier 1 according to the example and the protein-immobilized carrier according to the comparative example was performed according to the following procedure.
First, a 5% skim milk solution was added to each well of a 96-well plate on which the functional protein 50 was immobilized to block the antibody from nonspecific binding. Subsequently, 150 μL of the washing buffer attached to the above-mentioned ELISA Kit was added to each well and washed three times.

  Next, 50 μL of a biotinylated antibody adjusted to a concentration of 20 μg / mL was added to each well and reacted at 37 ° C. for 2 hours to bind the antibody to protein A. At this time, a total of 7 types of dilution series were prepared in which each biotinylated antibody was diluted from 1-fold to 1 / 30000-fold, and each was reacted with protein A. Subsequently, 200 μL of the washing buffer attached to the ELISA Kit was added to each well and washed three times to remove unbound antibodies.

  Subsequently, 50 μL of streptavidin-labeled peroxidase was added to each well and reacted at 37 ° C. for 1 hour to label the antibody bound to protein A with peroxidase. Subsequently, 200 μL of the washing buffer attached to the above ELISA Kit was added to each well and washed three times.

A peroxidase substrate was added to each well of the obtained 96-well plate for color development, hydrochloric acid was added to stop the reaction, and the efficiency of antibody-protein A binding was quickly confirmed.
The binding efficiency was confirmed by confirming the absorbance of each well of a 96-well plate using a microplate reader, and a wavelength of 480 nm was used as a detection wavelength. The result is shown in FIG.

FIG. 9 is a graph showing the efficiency of binding of the protein-immobilized carrier according to the example to the target substance.
In FIG. 9, the horizontal axis represents the concentration of the target substance (antibody) to be separated and purified by the protein-immobilized carrier, and the vertical axis represents the absorbance at 480 nm of the reaction solution in which peroxidase bound to the antibody reacted with the substrate. Represents. Moreover, the ● plot and solid line show the results with the protein-immobilized carrier according to the example, and the ◯ plot and broken line show the results with the protein-immobilized carrier according to the comparative example.
As shown in FIG. 9, in the protein-immobilized carrier 1 according to the example, an increase in absorbance was confirmed with respect to the protein-immobilized carrier according to the comparative example. That is, the plurality of proteins A carried on the protein-immobilized carrier 1 according to the example can interact with the antibody with higher efficiency as compared with the case of the protein-immobilized carrier according to the comparative example. It was confirmed that the protein-immobilized carrier 1 according to the example functions as a separation and purification material in which the binding efficiency with the target substance 60 is dramatically improved.
Therefore, in the protein-immobilized carrier in which the functional protein 50 is supported by being oriented in a predetermined direction like the protein-immobilized carrier 1 according to the embodiment, the functional protein 50 immobilized on the carrier 30 and the function It has been observed that the efficiency of binding with specific substances to which protein 50 specifically binds is improved.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described.

DESCRIPTION OF SYMBOLS 1 Protein immobilization support | carrier 3 Separation | purification apparatus 11 Culture solution receiving tank 12 Affinity column 13 Eluent tank 14 Equilibration liquid tank 15 Fraction tank 16 Drain 17 Filter 18 Eluate receiving tank 30 Carrier 40 Peptide linker 50 Functional protein 60 Target substance 70 Water-soluble carbodiimide

Claims (6)

A functional protein in which the N-terminal amino acid residue has a cysteine residue, a glycine residue or an alanine residue and has a function of specifically binding to a specific substance;
A peptide linker formed by linking one or more amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, tryptophan, methionine, glycine, threonine, histidine and cysteine;
A carrier carrying the functional protein via the peptide linker;
Having
The carrier and the N-terminus of the peptide linker are covalently bonded;
A protein-immobilized carrier, wherein the C-terminus of the peptide linker and the N-terminus of the functional protein are amide-bonded.   2. The protein-immobilized carrier according to claim 1, wherein the peptide linker has a length in which 3 to 200 amino acid residues are linked. A functional protein in which the N-terminal amino acid residue has an amino acid or cysteine to which a detachable modifying group containing a thiol group is bonded, and has a function of specifically binding to a specific substance; and
One or more amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, tryptophan, methionine, glycine, threonine, histidine and cysteine are linked, and the C-terminal carboxyl group Preparing a peptide linker in which is thioesterified;
A step of covalently bonding and fixing the N-terminus of the peptide linker prepared on a carrier having a plurality of carboxyl groups or amino groups on the surface;
Linking the N-terminus of the functional protein with an amide bond to the C-terminus of the prepared peptide linker;
A method for producing a protein-immobilized carrier comprising the steps of: The carrier has a plurality of carboxyl groups on the surface,
The method for producing a protein-immobilized carrier according to claim 3, wherein the covalent bond in the fixing step is formed by a carbodiimide compound.   The method for producing a protein-immobilized carrier according to claim 3 or 4, wherein the amide bond in the linking step is formed by a native chemical ligation method.   The protein according to any one of claims 3 to 5, further comprising a step of desulfurizing a thiol group of an N-terminal amino acid residue of the functional protein after the linking step. A method for producing an immobilization carrier.