JP2011168505A - Peptide having affinity for polycarbonate and/or polymethyl methacrylate, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a ligand peptide which can specifically or strongly binds to at least one of polycarbonates and polymethyl methacrylate. <P>SOLUTION: The ligand peptide contains a peptide comprising an amino acid sequence represented by one of sequence numbers 1 to 6, and has a mol.wt. of ≤10 kD. The peptide is bound to a desired protein, and can thereby specifically bind the protein to at least one of polycarbonates and polymethyl methacrylate. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a peptide that specifically binds to one or more of polycarbonate (hereinafter referred to as “PC” as appropriate) and polymethyl methacrylate (hereinafter referred to as “PMMA” as appropriate) (hereinafter referred to as “PC / PMMA affinity” as appropriate). Peptide)), and a typical method of using PC / PMMA affinity peptides.

  The present invention also relates to a method for efficiently screening peptides that specifically bind to a target substance such as polycarbonate or polymethyl methacrylate.

  In recent years, in the fields of medical diagnosis and biochemistry, research on protein chips in which functional proteins such as antibodies and enzymes are immobilized on plastic substrates has been actively conducted. In such a protein chip, there is a demand for a technique for stably and orientationally immobilizing proteins on a plastic substrate. As such a technique, a method is known in which an affinity peptide that specifically binds (adsorbs) to a plastic is introduced at the end of a functional protein. The present inventors introduce a peptide that specifically binds to polystyrene (referred to as “PS-tag”) at the end of the functional protein, and immobilize the functional protein into which the PS-tag has been introduced to polystyrene. As a result, it was confirmed that the functional protein can be immobilized stably and oriented (see, for example, Patent Document 1).

  As a peptide that specifically binds to plastics other than polystyrene, for example, Patent Document 2 discloses a peptide that specifically binds to polycarbonate, polystyrene, or polyurethane.

  Non-Patent Document 1 discloses a peptide that specifically binds to polymethyl methacrylate.

Conventionally, the phage display method has been used for screening of the conventionally known peptides. In this method, artificially produced peptides having a random amino acid sequence are displayed on the surface layer of filamentous phage, which is used as a peptide library, and a desired peptide is screened therefrom. An advantage of this method is that the library size that can be searched at one time is as large as -10 ¹² . However, the operation is very complicated, and there is a problem that it is often difficult to search for effective peptides due to the appearance of false positive clones due to non-specific adsorption of the phage itself.

International Publication No. 2009/101807 (International Publication Date: August 20, 2009) Special table 2005-518442 gazette (publication date: June 23, 2005)

Serizawa et al., `` Highly Specific Affinities of Short Peptides against Synthetic Polymers '', Langmuir 2007, 23, 11127-11133

  The above-mentioned conventionally known peptides can specifically bind to any of polycarbonates widely used as engineering plastics and polymethyl methacrylates used as substrates for protein chips, etc. Was not necessarily satisfactory enough. Further, a peptide that can specifically and firmly bind to both polycarbonate and polymethyl methacrylate has not been known. When a protein chip is prepared by immobilizing a functional protein on a substrate using a ligand peptide, the physiological activity of the functional protein may be inhibited or the three-dimensional structure may be changed depending on the peptide used. The use as a protein chip may be impaired. For this reason, a wide variety of ligand peptides are required.

  Therefore, the present invention provides a ligand peptide that can specifically and firmly bind to either or both of polycarbonate and polymethyl methacrylate, and is representative of a method for immobilizing proteins using the peptide. Provide usage.

  Furthermore, the present invention also provides a new efficient screening method replacing the phage display method as a screening method for peptides that specifically bind to a target molecule such as plastic.

  As a result of intensive studies to achieve the above object, the present inventors have developed a new screening method that replaces the phage display method, and using this screening method, either or both of polycarbonate and polymethyl methacrylate are developed. The present inventors have succeeded in finding a peptide capable of specifically binding to the present invention and completed the present invention.

That is, the ligand peptide according to the present invention is a ligand peptide used for specifically binding at least one of polycarbonate and polymethyl methacrylate by introducing the desired peptide into the desired protein, It includes the peptide shown in (a) or (b) and has a molecular weight of 10 kD or less.
(A) A peptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8.
(B) in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and polycarbonate and polymethyl methacrylate A peptide having an activity of specifically binding to at least one of the above.

The ligand peptide according to the present invention is a ligand peptide used for specifically binding the protein to both polycarbonate and polymethyl methacrylate, and comprises the peptide shown in the following (c) or (d): It may also be a ligand peptide characterized by having a molecular weight of 10 kD or less.
(C) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
(D) The amino acid sequence shown in SEQ ID NO: 4 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and specifically binds to polycarbonate and polymethyl methacrylate A peptide having the activity of

  The ligand peptide according to the present invention preferably has a molecular weight of 3.5 kD or less.

  The present invention also relates to a protein immobilization method in which a desired protein is specifically bound to and immobilized on at least one of polycarbonate and polymethyl methacrylate, and the ligand peptide according to the present invention is bound to the protein. An introduction step of introducing, and a contact step obtained by contacting the protein into which the ligand peptide is introduced and at least one of polycarbonate and polymethyl methacrylate obtained by the introduction step. Also includes immobilization methods.

  The introduction step in the protein immobilization method according to the present invention may be a step of producing a fusion protein of a desired protein and the ligand peptide according to the present invention.

  The present invention also provides a method for producing a protein that specifically binds to at least one of polycarbonate and polymethyl methacrylate, comprising an introduction step of introducing the ligand peptide according to the present invention into the protein. And a method for producing the protein.

  The introducing step in the method for producing a protein according to the present invention may be a step of producing a fusion protein of a desired protein and the ligand peptide according to the present invention.

  The present invention also includes a protein complex in which a protein produced by the protein production method according to the present invention is immobilized on a base material made of polycarbonate or polymethyl methacrylate.

  Furthermore, a protein chip in which a protein produced by the protein production method according to the present invention is immobilized on a substrate made of polycarbonate or polymethyl methacrylate is also included in the present invention.

  Further, the present invention is a method for screening a peptide that specifically binds to a target substance, wherein a primary selection step of selecting a protein that specifically binds to the target substance by contacting the protein library with the target substance, The peptide selected by the primary selection step is fragmented to prepare a peptide group, the peptide group prepared by the peptideization step is brought into contact with the target substance, and then the peptide group and the target substance are combined. A contact isolation step for isolation, and a secondary selection step for comparing peptides contained in the peptide group before and after the contact isolation step and selecting a peptide having a reduced content in the peptide group after the contact isolation step, The screening method is also included.

  The secondary selection step in the screening method according to the present invention is a step of comparing peptides contained in peptide groups before and after the contact isolation step and selecting peptides whose content in the peptide group is reduced by 70% or more after the contact isolation step. It is preferable.

  In the screening method according to the present invention, the target substance may be an organic polymer.

  In the present invention, the target substance may be one or more selected from the group consisting of polycarbonate and polymethyl methacrylate.

  Furthermore, in the present invention, the protein library may be composed of intracellular proteins.

  The ligand peptide according to the present invention is capable of specifically and firmly binding to polycarbonate and / or polymethyl methacrylate. Therefore, by introducing the peptide into a biomolecule such as a protein, a desired protein can be immobilized in a high density and orientation on at least one of a polycarbonate substrate and a polymethyl methacrylate substrate. Since the introduction position of the peptide according to the present invention can be arbitrarily controlled, it can be expected that the bioactivity of the biomolecule can be maintained. Therefore, the peptide according to the present invention can greatly contribute to the development of materials in the biochip and life science fields.

  Moreover, according to the screening method of the present invention, peptides capable of binding to various target substances such as polycarbonate and polymethyl methacrylate can be efficiently screened. Therefore, the screening method of the present invention can greatly contribute to immobilization of biomolecules such as proteins.

It is a figure for demonstrating one Embodiment of the screening method concerning this invention, and shows the process until it selects PC / PMMA affinity protein from the intracellular protein of colon_bacillus | E._coli. It is a figure for demonstrating one Embodiment of the screening method concerning this invention, and shows the process until it selects a PC / PMMA affinity peptide from PC / PMMA affinity protein. (A) A two-dimensional electrophoresis image of an adsorption sample containing intracellular proteins in E. coli, (b) an elution sample containing the protein eluted from the PC after contacting the adsorption sample with the PC It is a two-dimensional electrophoresis image. It is a SDS-PAGE image of PC / PMMA affinity protein (after purification) expressed in E. coli. It is a graph which shows the result of having measured the adsorption power with respect to PC and PMMA about PC / PMMA affinity protein. It is the chart figure which used the sample before and behind making the digestion product of BSA trypsin which is a control adsorb | suck to a PC piece. (A) is a chart of the sample before and after adsorbing the BIF tryptic digest on the PC piece, and (b) is the HPLC sample before and after adsorbing the BIF chymotrypsin digest on the PC piece. FIG. (A) is the chart which gave the sample before and behind adsorbing the tryptic digest of MLT to PC piece, and (b) is the sample before and after adsorbing the chymotrypsin digest of MLT to PC piece to HPLC. FIG. (A) is a chart of the sample before and after adsorbing the OMP tryptic digest on the PC piece, and (b) is the HPLC sample before and after adsorbing the OMP chymotrypsin digest on the PC piece. FIG. (A) is the chart which gave the sample before and behind adsorb | sucking the tryptic digest of ELN to PC piece, (b) is the HPLC before and after adsorbing the chymotrypsin digest of ELN to PC piece. FIG. It is a graph which shows the result of having measured the adsorption power with respect to PC of each PC / PMMA affinity peptide. It is a graph which shows the result of having measured the adsorption power with respect to PMMA of each PC / PMMA affinity peptide.

  An embodiment of the present invention will be described as follows. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range.

(1. Ligand peptide according to the present invention)
The ligand peptide according to the present invention is a peptide used to specifically bind at least one of polycarbonate (PC) and polymethyl methacrylate (PMMA) by introducing the peptide into a desired protein. .

  As used herein, the term “peptide” refers to a peptide in which two or more amino acids are linked by peptide bonds, and includes oligopeptides, polypeptides, proteins, homomeric peptides, and heteromeric peptides. In addition, the peptide may take an electrically neutral form or a salt form. The form consists of only the amino acid sequence in the sequence listing, or a fusion protein in which the peptide is fused to the end or inside of a specific protein. , Forms as a complex obtained by adding carbohydrate, polyethylene glycol, NCS group (isothiocyanate), NHS group (N-Hydroxy succinimide ester), maleimide group, thiol group, biotin, fluorescent dye, etc. Further, it may be in the form of a derivative or polymer obtained by cross-linking polymerization of a peptide by acetylation, amidation and / or multifunctional test.

  Here, the ligand peptide is a peptide comprising a peptide having an activity of specifically binding to one or both of PC and PMMA (referred to as “PC / PMMA affinity peptide”). Then, the protein is specifically bound to one or both of PC and PMMA via the ligand peptide by introducing the ligand peptide into a protein desired to be specifically bound to one or both of PC and PMMA. be able to. In this specification, “PC / PMMA” means “one or both of PC and PMMA” unless otherwise specified.

  The above ligand peptide is a protein chip prepared by fixing a functional protein such as an enzyme or an antibody on a substrate made of PC or PMMA, an immobilized enzyme, an affinity carrier in which an antibody is immobilized on a carrier, It can be preferably applied to immobilization of an antigen or an antibody in an ELISA method or the like.

  The protein into which the ligand peptide of the present invention is introduced is not particularly limited, and can be applied to, for example, an antigen, an antibody, a lectin, an enzyme, or a receptor protein. More specifically, Glutathione S-Transferase (GST), maltose binding protein (MBP), alkaline phosphatase (ALP), peroxidase (POD), luciferase, green fluorescent protein (GFP), β-galactosidase (β -Gal), trypsin, chymotrypsin, thrombin, Factor Xa, angiotensin converting enzyme, tyrosine kinase, insulin receptor, EGF receptor, streptavidin (SA), monoclonal antibody (Mab), polyclonal antibody (Pab), single chain antibody (scFv) ), Multivalent single chain antibodies (sc (Fv) n) (eg, bivalent single chain antibodies (sc (Fv) 2)), constant region fusion single chain antibodies (scFv-Fc), Fab fragments and F (ab ') 2 fragment (antibody fragment containing the antigen-binding site), complement system protein C1q, concanavalin (ConA), lentil lectin (LCA), it is possible to apply an antibody binding protein (Protein A, ZZ, Protein G, Protein L, etc.) or the like.

  As described above, since the ligand peptide is preferably used for immobilizing functional proteins such as enzymes and antibodies as described above, the molecular weight needs to be 10 kD or less so as not to inhibit the function of the functional protein. When the molecular weight of the ligand peptide exceeds 10 kD, the function (physiological activity) of the protein to be immobilized is impaired, the solubility of the introduced protein is decreased, or the molecular size of the protein becomes too large, so that the protein is immobilized at high density. In many cases. Therefore, it is preferable that the molecular weight of the ligand peptide be as small as possible within a range having an activity of specifically binding to one or both of PC and PMMA. For example, the molecular weight of the ligand peptide is more preferably 10 kD or less, and most preferably 3.5 kD or less.

  Here, “having an activity to specifically bind to one or both of PC and PMMA” means that the selection step in the screening method according to the present invention described later satisfies the criteria to be selected. .

  Examples of the peptide constituting the ligand peptide according to the present invention (“PC / PMMA affinity peptide”) include a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.

  A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (referred to as “PCMLT10”) has an activity of specifically binding to a PC / PMMA affinity protein (PC or PMMA or both) according to the study by the present inventors. E. coli-derived Malto porin (abbreviated as “MLT”) identified as The amino acid sequence and base sequence of MLT are shown in Sequence Listings 17 and 18, respectively. Information on the amino acid sequence and base sequence of MLT is available from NCBI Gene Bank (http://www.ncbi.nlm.nih.gov/Genbank/) (Accession No. CP001509 REGION: 4153898-4155238) . The molecular weight, the position of PCMLT10 in the MLT total amino acid sequence, the amino acid sequence, and the secondary structure are shown in Table 2 below. This PCMLT10 is a peptide having an activity that specifically binds to PC. The secondary structure of PCMLT10 is characterized by containing many α-helix structures. An example of the base sequence of DNA encoding PCMLT10 is not particularly limited, but an example is the base sequence shown in SEQ ID NO: 9. Other codons may be used as long as the base sequence encodes PCMLT10.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (referred to as “PCMLT8”) is a peptide found from E. coli-derived MLT identified as a PC / PMMA affinity protein by the inventors' investigation. The molecular weight, the position of PCMLT8 in the entire amino acid sequence of MLT, the amino acid sequence, and the secondary structure are shown in Table 2 below. PCMLT8 is a peptide having an activity that specifically binds to PC. The secondary structure of PCMLT8 is characterized by containing a lot of β-sheet structures. The base sequence of DNA encoding PCMLT8 is not particularly limited, and examples thereof include the base sequence shown in SEQ ID NO: 10. Other codons may be used as long as the base sequence encodes PCMLT8.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (referred to as “PCOMP6”) is seen from OmpF porin (abbreviated as “OMP”) derived from Escherichia coli identified as a PC / PMMA affinity protein by the present inventors' investigation. It is a released peptide. The amino acid sequence and base sequence of OMP are shown in Sequence Listings 19 and 20, respectively. Information on the amino acid sequence and base sequence of OMP is available from NCBI Gene Bank (http://www.ncbi.nlm.nih.gov/Genbank/) (Accession No. CP001509 REGION: 990984-992072) . The molecular weight, the position of PCOMP6 in the entire amino acid sequence of OMP, the amino acid sequence, and the secondary structure are shown in Table 3 below. PCOMP6 is a peptide having an activity of specifically binding to PC. The secondary structure of PCOMP6 is characterized by containing a large amount of β-sheet structure. The base sequence of DNA encoding PCOMP6 is not particularly limited, and examples thereof include the base sequence represented by SEQ ID NO: 11. Other codons may be used as long as the base sequence encodes PCOMP6.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (referred to as “PCOMP3”) is a peptide found from EMP derived from E. coli identified as a PC / PMMA affinity protein by the inventors' investigation. Its molecular weight, position of PCOMP3 in the entire amino acid sequence of OMP, amino acid sequence, and secondary structure are shown in Table 3 below. PCOMP3 was the same peptide as PMOMP21 shown in Table 5 below. That is, this PCOMP3 (PMOMP21) is a peptide having an activity of specifically binding to PC and PMMA. The secondary structure of PCOMP3 (PMOMP21) is characterized by containing a lot of β-sheet structures. The base sequence of DNA encoding PCCOMP3 (PMOMP21) is not particularly limited, and examples thereof include the base sequence shown in SEQ ID NO: 11. Other codons may be used as long as the base sequence encodes PCOMP3.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 (referred to as “PCOMP7”) is a peptide found from EMP derived from E. coli that has been identified as a PC / PMMA affinity protein by the inventors' investigation. The molecular weight, position of PCOMP7 in the entire amino acid sequence of OMP, amino acid sequence, and secondary structure are shown in Table 3 below. This PCOMP7 is a peptide having an activity that specifically binds to PC. The secondary structure of PCOMP7 is characterized by being composed of an α-helix structure and a β-sheet structure. The base sequence of DNA encoding PCOMP7 is not particularly limited, and examples thereof include the base sequence represented by SEQ ID NO: 13. Other codons may be used as long as the base sequence encodes PCOMP7.

  A peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 (referred to as “PCELN8”) is derived from Elongation factor Tu (“ELN”) derived from E. coli that has been identified as a PC / PMMA affinity protein by the present inventors. It is a found peptide. The molecular weight, the position of PCELN8 in the entire ELN amino acid sequence, amino acid sequence, and secondary structure are shown in Table 4 below. This PCELN8 is a peptide having an activity that specifically binds to PC. The secondary structure of PCELN8 is characterized by being composed only of a β-sheet structure. The base sequence of DNA encoding PCELN8 is not particularly limited, and examples thereof include the base sequence shown in SEQ ID NO: 14. As long as the base sequence encodes PCELN8, other codons may be used. The amino acid sequence and base sequence of ELN are shown in Sequence Listings 21 and 22, respectively. Information on the amino acid sequence and base sequence of ELN is available from NCBI Gene Bank (http://www.ncbi.nlm.nih.gov/Genbank/) (Accession No. CP001509 REGION: 4082550-4083734) .

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 (referred to as “PMOMP19”) is a peptide found from EMP derived from E. coli that was identified as a PC / PMMA affinity protein by the inventors' investigation. The molecular weight, position of PMOMP19 in the entire amino acid sequence of OMP, amino acid sequence, and secondary structure are shown in Table 5 below. PMOMP19 is a peptide having an activity that specifically binds to PMMA. The secondary structure of PMOMMP 19 is characterized by being mainly composed of a β-sheet structure and a coil structure. The base sequence of DNA encoding PMOMP19 is not particularly limited, and for example, the base sequence shown in SEQ ID NO: 15 can be mentioned.

  A peptide consisting of the amino acid sequence shown in SEQ ID NO: 8 (referred to as “PMOMP25”) is a peptide found from EMP derived from E. coli identified as a PC / PMMA affinity protein by the study of the present inventors. The molecular weight, the position of PMOMP25 in the entire amino acid sequence of OMP, the amino acid sequence, and the secondary structure are shown in Table 5 below. PMOMP25 is a peptide having an activity that specifically binds to PMMA. The secondary structure of PMOMMP25 is characterized by an α-helix structure and a β-sheet structure. The base sequence of DNA encoding PMOMP25 is not particularly limited, and examples thereof include the base sequence represented by SEQ ID NO: 16.

  Moreover, the ligand peptide of this invention should just contain the said PC / PMMA affinity peptide, and may consist only of PC / PMMA affinity peptide. In addition, the ligand peptide of the present invention may be one in which other amino acids are added to the PC / PMMA affinity peptide as long as the molecular weight as a whole of the ligand peptide is 10 kD or less. For example, the ligand peptide may be one in which an amino acid necessary for linking a protein to be bound to PC / PMMA and the ligand peptide is added to the PC / PMMA affinity peptide.

  The ligand peptide of the present invention is an amino acid present on the C-terminal side or N-terminal side of the PC / PMMA affinity peptide in the amino acid sequence of the PC / PMMA affinity protein from which each PC / PMMA affinity peptide is derived. May be added to the PC / PMMA affinity peptide.

  Specifically, PCMLT10 corresponds to amino acids at positions 431 to 457 of the entire amino acid sequence of MLT (SEQ ID NO: 17), but the ligand peptide may include amino acids located before and after that. Good. Although it does not specifically limit, For example, the ligand peptide which consists of the 420th-460th position of sequence number 17 may be sufficient.

  Similarly, PCMLT8 corresponds to amino acids at positions 341 to 360 of the entire amino acid sequence of MLT (SEQ ID NO: 17), but the ligand peptide may include amino acids located before and after that. Although it does not specifically limit, For example, the ligand peptide which consists of the 340th-365th position of sequence number 17 may be sufficient.

  Similarly, PCOMP6 corresponds to amino acids at positions 163 to 182 of the entire amino acid sequence of OMP (SEQ ID NO: 19), but the ligand peptide may include amino acids located before and after that. Although it does not specifically limit, For example, the ligand peptide which consists of the 160th-185th position of sequence number 19 may be sufficient.

  Similarly, PCOMP3 (PMOMP19) corresponds to the amino acids at positions 276 to 299 of the entire amino acid sequence of OMP (SEQ ID NO: 19), but the ligand peptide may include amino acids located before and after that. Good. Although it does not specifically limit, For example, the ligand peptide which consists of the 270th-300th position of sequence number 19 may be sufficient.

  Similarly, PCOMP7 corresponds to amino acids 5 to 28 of the entire amino acid sequence of OMP (SEQ ID NO: 19), but the ligand peptide may include amino acids located before and after that. Although it does not specifically limit, For example, the ligand peptide which consists of 1st-30th position of sequence number 19 may be sufficient.

  Similarly, PCELN8 corresponds to amino acids at positions 125 to 137 of the entire amino acid sequence of ELN (SEQ ID NO: 21), but the ligand peptide may include amino acids located before and after that. Although it does not specifically limit, For example, the ligand peptide which consists of 120-140th position of sequence number 21 may be sufficient.

  Similarly, PMOMP19 corresponds to amino acids at positions 191 to 218 of the entire amino acid sequence of OMP (SEQ ID NO: 19), but the ligand peptide may include amino acids located before and after that. Although it does not specifically limit, For example, the ligand peptide which consists of the 190th-220th position of sequence number 19 may be sufficient.

  Similarly, PMOMP25 corresponds to amino acids 304 to 327 of the entire amino acid sequence of OMP (SEQ ID NO: 19), but the ligand peptide may include amino acids located before and after that. Although it does not specifically limit, For example, the ligand peptide which consists of the 300th-330th position of sequence number 19 may be sufficient.

  The PC / PMMA affinity peptide constituting the ligand peptide of the present invention is not limited to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6, and in SEQ ID NOs: 1 to 1, one or several A peptide having an amino acid sequence in which amino acids are substituted, deleted, inserted, and / or added, and having an activity of specifically binding to at least one of polycarbonate and polymethyl methacrylate (“mutant peptide”). May be. That is, in the present invention, a modified amino acid sequence shown in SEQ ID NOs: 1 to 6 is allowed as long as it has an activity of specifically binding to PC / PMMA. Here, the above “one or several amino acids are substituted, deleted, inserted, and / or added” means substitution, deletion, by a known mutant protein production method such as site-directed mutagenesis. It means that a sufficient number of amino acids (preferably 5 or less, more preferably 3 or less) that can be inserted and / or added are substituted, deleted, inserted and / or added. It is naturally possible for those skilled in the art to produce a mutant peptide (or a ligand peptide comprising the mutant peptide). What is necessary is just to judge whether the mutant peptide produced in this way (or a ligand peptide comprising the mutant peptide) has an activity of specifically binding to PC / PMMA.

  The ligand peptide according to the present invention can be produced by a known genetic engineering technique or chemical synthesis method. For example, when using genetic engineering techniques, a DNA encoding a ligand peptide is prepared and inserted into a vector capable of autonomous replication to obtain a recombinant DNA, which is transformed into E. coli, Bacillus subtilis, actinomycetes, yeast, filamentous form. A desired ligand peptide can be collected from the culture by introducing it into an appropriate host such as a fungus, plant cell, insect cell, animal cell or the like to obtain a transformant. Further, a DNA encoding the ligand peptide according to the present invention can be prepared, and a desired ligand peptide can be synthesized by a cell-free protein synthesis system using wheat germ, Escherichia coli cell extract or the like.

On the other hand, using a conventional peptide chemical synthesis method such as “solid phase method” or “liquid phase method”, a desired ligand peptide is synthesized by sequentially dehydrating and condensing the ligand peptide according to the present invention. can do. Examples of known condensation methods and protecting group removal include the methods described in the following references (i) to (v).
(I) M.M. Bodanszky and M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Yajima Haruaki, Development of follow-up medicines Volume 14 Peptide synthesis Hirokawa Shoten The ligand peptides obtained above can be obtained by conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. It can be purified and isolated as appropriate in combination. In addition, since the ligand peptide acquired by the above has a PC / PMMA affinity peptide, a ligand peptide is made into PC / PMMA by making the solution containing the product from a transformant contact PC / PMMA surface. It can be directly adsorbed on the surface of PMMA for easy separation and purification. The solution containing the product refers to all solutions containing a desired ligand peptide and containing unnecessary contaminants originating from the host, and can be obtained by, for example, centrifuging a cell disruption solution or cell disruption solution. Soluble fraction, solubilized insoluble fraction obtained by centrifuging cell disruption fluid, cell membrane fraction, cell wall fraction, secreted product secreted from cells, body fluid, or incomplete of these Including purified products.

  Furthermore, when a heterologous gene is expressed in a large amount in a host by introducing a recombinant DNA, an inclusion body (inclusion body) that is an insoluble and inactive aggregate is used to avoid adverse effects on the host by the produced protein. However, even if the ligand peptide according to the present invention is produced as an inclusion body, the inclusion body is solubilized with a denaturing agent and then immobilized on the PC / PMMA surface as it is. In addition, the ligand peptide according to the present invention may be refolded by removing the denaturant in an immobilized state.

  In addition, the ligand peptide according to the present invention can be immobilized on the PC / PMMA surface as it is, even if the steric structure is modified for some reason, not only for inclusion bodies. In some cases, the ligand peptide according to the present invention can be refolded by adding an appropriate refolding buffer. Causes of denaturation include physical causes such as heating, freezing, high pressure, ultrasonic waves, ultraviolet rays, X-rays, agitation, adsorption, dilution, extreme acidic or alkaline, organic solvents, heavy metal salts, denaturing agents, surface activity There are chemical causes such as chemicals.

(2. Protein immobilization method according to the present invention)
The protein immobilization method according to the present invention is a method in which a desired protein is specifically bound to and immobilized on at least one of polycarbonate and polymethyl methacrylate. An introduction step of introducing the protein into the protein, and a contact step obtained by contacting the protein into which the ligand peptide is introduced and at least one of polycarbonate and polymethyl methacrylate obtained by the introduction step. .

  As described above, since the ligand peptide according to the present invention is a peptide comprising a PC / PMMA affinity peptide having an activity of specifically binding to PC / PMMA, the ligand peptide is introduced into a desired protein. By doing so, the protein can be specifically bound to PC / PMMA via a ligand peptide. In addition, since the ligand peptide according to the present invention has high affinity with PC / PMMA, it is possible to immobilize proteins with high density and strength. Furthermore, by controlling the position where the ligand peptide is introduced into the protein, the desired protein can be immobilized in an oriented manner, and the merit that it can be immobilized while maintaining the physiological activity of the protein can be enjoyed.

[Introduction process]
The introduction step included in the protein immobilization method according to the present invention is not particularly limited as long as it is a step of introducing a ligand peptide into a desired protein. For example, the introduction step can be performed using, for example, a known genetic engineering technique or a peptide chemical synthesis method. The desired protein into which the ligand peptide is introduced is as described above.

  The site for introducing the ligand peptide is preferably a site that does not interfere with the physiological activity inherent in the desired protein, for example, other than an antigenic determinant in the case of an antigen, and other than an antibody active group in the case of an antibody. In particular, since it is preferable to immobilize on PC / PMMA while maintaining the three-dimensional structure of the desired protein, it is preferable to introduce the ligand peptide at a site near the C-terminal or N-terminal of the desired protein. In addition to the ligand peptide, a labeling affinity tag or the like may be separately introduced into the desired protein.

  In particular, when a protein having a variable region in which an antigen-binding site is formed as a desired protein, the protein is located on the C-terminal side of the heavy chain variable region or on the C-terminal side (including both) of the light chain variable region. Introduce ligand peptide. The C-terminal side of the variable region refers to all positions from the C-terminus of the heavy chain or light-chain variable region to the C-terminus of the entire peptide that do not inhibit antigen binding of the variable region.

When the desired protein is an antibody molecule, the constant region (C _H 1, C _H 2, C _H 3, C _H 4) located on the C-terminal side of the variable region (V _H ) of the heavy chain (H chain) It is preferably introduced at one or a plurality of positions at the N-terminal or C-terminal in the domain of N, or at the N-terminal or C-terminal in the constant region (C _L ) of the light chain (L chain). It is preferably introduced into one or a plurality of positions of the N-terminal, C-terminal, and C-terminal of the light chain in the domain of the constant region (C _H 2, C _H 3, C _H 4).

In the case of Fab fragment or F (ab ′) ₂ fragment, the C-terminal in the variable domain (V _H and V _L ) of the heavy chain or the light chain, and the N-terminal in the domain of the constant domain (C _H 1 and C _L ) Or at the C-terminal or at one or more locations, and particularly at one or more locations at the C-terminus of the heavy chain or the C-terminus of the light chain.

Peptides that bind heavy and light chains, such as single chain antibodies (scFv), multivalent single chain antibodies (sc (Fv) _n ), and constant region fusion single chain antibodies (scFv-Fc) In the case of having (linker peptide), a ligand peptide may be introduced between the linker peptide and the heavy chain or light chain, or inside the linker peptide. Here, the linker peptide links one C-terminus of the heavy chain or light chain and the other N-terminus, but the entire linker peptide, that is, the heavy chain is located on the C-terminal side of the heavy chain or light chain. Alternatively, the region from the portion bonded to the C-terminus of the light chain to the other N-terminus is also included. A labeling affinity tag other than the present amino acid sequence may be separately introduced between the linker peptide and the heavy or light chain, or inside the linker peptide. In the case of a single chain antibody (scFv), the light chain variable region (V _L ) or the light chain variable region including the inside of the linker peptide from the heavy chain variable region (V _H ) C-terminal side (V _H ) The ligand peptide is introduced into one or both between V _L ) up to the N-terminus.

By introducing a ligand peptide into a plurality of locations, the three-dimensional structure of the peptide can be maintained in a more normal state. For example, a ligand peptide is introduced into the C-terminal side of the heavy chain and the light chain of the Fab fragment or F (ab ′) ₂ fragment, respectively, and the C-terminal side of the heavy chain and light chain of the single-chain antibody (scFv), respectively. If a ligand peptide is introduced, the site becomes easy to bind to the PC / PMMA surface, so that the antigen-binding site faces outward and an antigen-antibody reaction is likely to occur.

  When the introduction step is performed using a genetic engineering technique, a polynucleotide (DNA) encoding the ligand peptide according to the present invention is linked to a polynucleotide (DNA) encoding a desired protein by a known method, and then linked. The protein may be expressed by introducing an expression vector containing the polynucleotide (DNA) into the host cell. That is, the introduction step can be performed by preparing a fusion protein of a ligand peptide and a desired protein. A nucleotide (nucleotide chain) necessary for linking a polynucleotide (DNA) encoding a desired protein and a polynucleotide (DNA) encoding a ligand peptide may be added between the two. At this time, it is necessary to add nucleotides (nucleotide chains) that can be translated into amino acids corresponding to the desired protein and ligand peptide.

  The specific type of vector that can be used in the introduction step of the present invention is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide according to the present invention, and a vector in which this and the polynucleotide according to the present invention are incorporated into various plasmids is used as an expression vector. Use it.

  Examples of the vector include E. coli-derived plasmids (eg, pBR322, pBR325, pUC18, pUC118), animal viruses such as retrovirus, vaccinia virus, and baculovirus.

  The promoter that can be used in the present invention may be any promoter that is suitable for the host used for gene expression. For example, when the host is Escherichia coli, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc. are used. When animal cells are used as the host, SRα promoter, SV40 promoter, LTR promoter, CMV Examples include promoters and HSV-TK promoters.

  In addition, an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 origin of replication and the like known in the art can be added to the vector that can be used in the present invention as desired.

  In addition, the vector that can be used in the present invention preferably contains at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline resistance gene or ampicillin resistance gene for culture in E. coli and other bacteria. By using the above selectable marker, it can be confirmed whether or not the vector has been introduced into the host cell, and further whether or not the vector is reliably expressed in the host cell. Alternatively, the protein of the present invention may be expressed as a fusion polypeptide. For example, a green fluorescent protein GFP (Green Fluorescent Protein) derived from Aequorea jellyfish is used as a marker, and a fusion peptide of a ligand peptide, a desired protein, and GFP is used. It may be expressed as a peptide.

  The host cell applied in the present invention is not particularly limited, and various conventionally known cells such as Escherichia coli, insect cells and animal cells can be preferably used. Specifically, for example, bacteria such as Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), and Xenopus laevis mothers such as Xenopus laevis However, it is not particularly limited.

  Specific examples of Escherichia coli include those used in Examples described later, Escherichia coli K12 / DH1 (reference: Proc. Natl. Acad. Sci. USA, 60: 160 (1968)), JM103 (reference: Nucleic Acids Research, 9: 309 (1981)), JA221 (references: Journal of Molecular Biology, 120: 517 (1978)), and HB101 (references: Journal of Molecular Biology 9, 19:45). Used.

  As animal cells, for example, monkey cell COS-7, Vero, Chinese hamster cell CHO, mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL cell and the like are used. Appropriate culture media and conditions for the above-described host cells are well known in the art.

  A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. . For example, when a fusion protein of a ligand peptide and a desired protein is transferred and expressed in an insect, an expression system using a baculovirus may be used.

[Contact process]
The contact process included in the protein immobilization method according to the present invention is limited as long as it is a contact process in which a protein into which a ligand peptide is introduced is brought into contact with at least one of PC and PMMA. Not a thing. For example, when a protein is immobilized on a substrate made of PC or PMMA, a solution prepared by dissolving the protein into which the ligand peptide is introduced in an appropriate solvent is dropped onto the surface of the substrate made of PC or PMMA. Good. After dropping the solution, the solution is allowed to stand for an appropriate time, and then the surface of the substrate may be washed with an appropriate buffer solution (PBS or the like) or water in order to remove unadsorbed protein.

  When the protein is immobilized on a carrier made of PC or PMMA, the carrier may be suspended in a protein solution into which the ligand peptide has been introduced. In this case, it is preferable to stir the suspension by a stirring means such as a magnetic stirrer because the contact efficiency between the protein into which the ligand peptide is introduced and the carrier is increased.

  The concentration, contact time, contact temperature, and the like of the protein into which the ligand peptide is introduced used in the contact step may be adopted after considering suitable conditions depending on the type of protein.

  In addition, the contact step is [4. It can be carried out with reference to the description of [Measurement of Adsorption Power of Affinity Peptide]. Note that 2% DMSO + PBS can be used for preparing the PCOMP3 (PMOMP21) solution, 2% DMSO + PBS can be used for preparing the PCOMP6 solution, and 1% DMSO + PBS can be used for preparing the PCOMP7 solution. 1% DMSO + PBS can be used for PCMLT8 solution preparation, 0.8M urea + PBS can be used for PCMLT10 solution preparation, and 0.08M urea + can be used for PCELN8 solution preparation. PBS can be used, 0.8M urea + PBS can be used for preparing a solution of PMOMP19, and 0.8M urea + PBS can be used for preparing a solution of PMOMP25.

  In addition, although the aspect which performs the contact process using the solution containing the protein in which the ligand peptide was introduced was described above, the contact between the protein into which the ligand peptide was introduced and PC / PMMA may be performed in the gas phase. .

(3. Method for producing a protein that specifically binds to PC / PMMA according to the present invention)
A method for producing a protein according to the present invention is a method for producing a protein that specifically binds to at least one of polycarbonate and polymethyl methacrylate, wherein the above-described ligand peptide according to the present invention is introduced into the protein. It is characterized by including a process. That is, the protein production method according to the present invention is the same as the “introduction step” described in the above section (2. Protein immobilization method according to the present invention). Therefore, the description of the “introduction step” in (2. The method for immobilizing a protein according to the present invention) can be used for the description of the protein production method according to the present invention.

(4. Protein complex and protein chip according to the present invention)
The protein complex according to the present invention is a protein complex in which a protein produced by the method for producing a protein that specifically binds to PC / PMMA according to the present invention is immobilized on a substrate made of PC / PMMA. It is. In other words, the protein complex according to the present invention is a complex of a protein and a base material in which a desired protein is immobilized on a substrate made of PC / PMMA by the protein immobilization method according to the present invention. . Therefore, the protein chip manufacturing method according to the present invention can use the description of the “introduction step” in (2. Protein immobilization method according to the present invention).

  The protein complex according to the present invention can be applied to a packing material for affinity chromatography, an analytical instrument such as a microtiter plate or a protein chip used in an ELISA method or the like. A plate or film in which protein is immobilized is particularly called “protein chip”. Protein chips are often used for antibody and antigen screening, drug development, and the like.

  As described above, since the ligand peptide according to the present invention is a peptide comprising a PC / PMMA affinity peptide having an activity of specifically binding to PC / PMMA, the ligand peptide is introduced into a desired protein. By doing so, the protein can be specifically bound to PC / PMMA via a ligand peptide. In addition, since the ligand peptide according to the present invention has high affinity with PC / PMMA, it is possible to immobilize proteins with high density and strength. Furthermore, by controlling the position where the ligand peptide is introduced into the protein, the desired protein can be immobilized in an oriented manner, and the merit that it can be immobilized while maintaining the physiological activity of the protein can be enjoyed. In particular, immobilization in a state where physiological activity is maintained in a protein chip or a microtiter plate is important. In addition, high density immobilization of protein chips contributes to improvement of analysis efficiency.

  As used herein, the term “substrate” is intended to mean a substance capable of carrying an object (polypeptide or protein), and is used interchangeably with the term “support”. Such a base material (support) is not limited as long as the portion on which the protein is immobilized is composed of at least PC / PMMA, and the entire substrate is not necessarily composed of PC / PMMA. For example, although it may be a substrate made of only PC / PMMA, PC / PMMA is laminated or coated on a base material made of other materials (glass, metal, organic polymer other than PC and PMMA, etc.). It may be a substrate. When PC / PMMA is laminated or coated on other materials, at least a portion where the protein is to be immobilized needs to be laminated or coated with PC / PMMA, and the entire substrate may not be laminated or coated.

  Although the shape of a base material is not specifically limited, For example, board | substrates, such as plate shape and a film form, a microtiter plate, bead shape (granular form), a fiber form, etc. are mentioned.

In order to make the protein easier to fix, it is possible to use a material that has been subjected to a hydrophilic treatment by altering the surface of the substrate. Usually, PC / PMMA is hydrophobic, but by performing various hydrophilic treatments on this surface, a substrate having a hydrophilic surface can be obtained. For example, the surface of the substrate can be hydrophilized by performing UV + O ₃ treatment or plasma oxidation treatment on the surface of PC / PMMA.

(5. Screening method according to the present invention)
The screening method according to the present invention is a method for screening a peptide that specifically binds to a target substance, wherein a protein library and a target substance are brought into contact with each other to select a protein that specifically binds to the target substance. The peptide group selected by the selection process, the protein selected by the primary selection process, the peptideization process for preparing the peptide group, the peptide group prepared by the peptideization process and the target substance are contacted, and then the peptide group and the target A contact isolation step for isolating the substance, and a secondary selection step for comparing peptides contained in the peptide group before and after the contact isolation step and selecting a peptide having a reduced content in the peptide group after the contact isolation step. A screening method characterized.

  Here, the “target substance” is not particularly limited as long as it is a substance that desires to find a peptide that specifically binds to the target substance. However, in the selection process and the contact isolation process, since it is preferable from the viewpoint of contact efficiency that the target substance is contacted with the protein library or peptide group in the liquid phase, the target substance is subjected to the selection process or the contact isolation process. It is preferably a substance that can be solid in the liquid phase.

  The target substance may be an inorganic substance or an organic substance. For example, common plastics (eg, polystyrene, polyurethane, PC, PMMA), silicones, synthetic polymers, metals (including mixed metal alloys), metal oxides (eg, glass), non-metal oxides, ceramics, etc. Can be mentioned.

  A typical synthetic polymer that can be a target substance is not particularly limited, but polytetrafluoroethylene, expanded polytetrafluoroethylene, GORE-TEX (registered trademark), polytetrafluoroethylene, fluorinated ethylene propylene, Hexafluoropropylene, polymethylmethacrylate (PMMA), pelletane (commercial polyurethane, PELL), 2-hydroxyethyl methacrylate (PHEMA), polyethylene terephthalate (PET), polyethylene, polypropylene, nylon, polyethylene terephthalate, polyurethane, silicone Rubber, polystyrene, polysulfone, polyester, polyhydroxy acid, polycarbonate, polyimide, polyamide, polyamino acid, and these The combination of is mentioned. In one embodiment, the synthetic polymer comprises a foamed polymer or a porous polymer. In another embodiment, the synthetic polymer comprises a nylon structure.

  Representative metals that can be target materials include, but are not limited to, titanium, stainless steel, gold, silver, rhodium, zinc, platinum, rubidium, and copper. Examples of ceramic materials that can be used as target substances include silicone oxide, aluminum oxide, alumina, silica, hydroxyapatite, glass, quartz, calcium oxide, calcium phosphate, indium tin oxide (ITO), polysilanol, phosphorus oxide, and combinations thereof. For example, but not limited to.

  Here, “protein library” means a group consisting of a plurality of types of proteins. However, “protein library” in the present invention means a phage library that displays random peptides in the phage display method. Such a protein library may exist in the form of a solution or in the form of a solid, but is preferably present in the form of a solution from the viewpoint of contact efficiency with a target substance in the selection process or the contact isolation process.

  The origin of the protein library is not particularly limited as long as it includes a plurality of types of proteins. For example, cell extracts or cell disruptions containing intracellular proteins, cell secretions, serum, plasma, Examples thereof include body fluids and food extracts.

  One embodiment of the screening method according to the present invention will be specifically described with reference to FIGS. 1 and 2, but the present invention is not limited to this. In the following description, PC / PMMA was used as the target substance, and E. coli cell lysate (E. coli intracellular protein) was used as the protein library.

[Primary selection process]
The primary selection step is a step of selecting a protein that specifically binds to the target substance by bringing the protein library into contact with the target substance. FIG. 1 shows an outline of the primary selection process. First, E. coli intracellular protein solution (E. coli cell extract) is brought into contact with PC / PMMA. The contact may be carried out by dropping (coating) the E. coli cell extract on the surface of the PC / PMMA piece, or the PC / PMMA piece (PC / PMMA plate fragment, PC / PMMA particles etc.) may be added to bring them into contact. When PC / PMMA is added and brought into contact with the E. coli cell extract, it is preferable to stir the E. coli cell extract containing PC / PMMA. About stirring conditions, what is necessary is just to employ | adopt after examining suitably conditions according to protein concentration, the addition amount of PC / PMMA, etc. suitably.

  After contacting the E. coli cell extract with PC / PMMA for an appropriate time, the surface of PC / PMMA is washed with water or a buffer solution (PBS, TBS, HBS, etc.) or a surfactant such as Tween 20 or Triton X-100. Wash with an appropriate washing solution such as a buffer solution. By this operation, proteins not bound to PC / PMMA are removed. For example, in this washing operation, the E. coli cell extract containing the PC / PMMA piece is subjected to solid-liquid separation by centrifugation, the washing solution is added to the obtained precipitate, suspended, and then centrifuged again to collect the precipitate. It can be implemented more than that. Moreover, you may perform the said solid-liquid separation by filtration. The cleaning operation may be performed a plurality of times.

  On the PC / PMMA recovered by the washing operation, there is a protein specifically bound to PC / PMMA. Such proteins are eluted from PC / PMMA using a suitable eluent. The eluent that can be used in the present invention is not particularly limited. For example, protein denaturants such as urea, guanidine hydrochloride, and SDS, acids, bases, salts, chaotropic salts such as sodium thiocyanate, and monomer molecules. Or a solution such as an organic solvent. In examples described below that could be used, Lysis Buffer (8M urea, 4% CHAPS, 2v / v% Pharmalyte pH 3-10, 1% DTT) was used. However, the present invention is not limited to this, and a preferable eluate can be appropriately employed depending on the type of the target substance.

  The protein recovered by the above operation is a protein that can specifically adsorb to PC / PMMA (“PC / PMMA affinity protein”).

[Peptidation step]
The peptidization step is a step of preparing a peptide group by fragmenting the PC / PMMA affinity protein selected in the primary selection step. In other words, the peptideization step is a step of preparing a group consisting of peptides constituting the protein by cleaving the PC / PMMA affinity protein. FIG. 2 shows an outline of the peptideization step.

  A proteolytic enzyme (protease, expressed as “digestive enzyme” in FIG. 2) capable of degrading a protein into a peptide chain of an appropriate length can be preferably used for such a peptide formation step. As the proteolytic enzyme, trypsin, chymotrypsin, papain, pepsin, Factor Xa, Thrombin, Lys-C, Arg-C, Asp-N, V8 and the like can be preferably used. In addition to the method using the above protein enzyme, the peptidation step is performed by CNBr treatment, Formic Acid treatment, heat treatment, autoclave treatment, ultraviolet irradiation, supercritical water treatment, subcritical water treatment, hydrogen peroxide-electrolysis treatment, etc. Can also be done.

  In the case of carrying out the peptidation step using a proteolytic enzyme, the amount of enzyme used may be appropriately selected according to the concentration of the protein to be degraded. Moreover, about reaction conditions, such as reaction temperature and reaction pH, suitable conditions can be set suitably according to the kind of enzyme.

[Contact isolation process]
The contact isolation step is a step of isolating the peptide group and the target substance after contacting the peptide group prepared by the above-mentioned peptide formation step with the target substance PC / PMMA. FIG. 2 shows an outline of the contact isolation process.

  The contact isolation step can be performed, for example, as follows. First, a solution containing a peptide group (peptide group solution) is brought into contact with PC / PMMA. The contact may be performed by dropping (coating) the peptide group solution on the surface of the PC / PMMA piece, or the PC / PMMA piece (a piece of PC / PMMA plate, PC / PMMA plate in the peptide group solution). Particles etc.) may be added to bring them into contact. When PC / PMMA is added and brought into contact with the peptide group solution, it is preferable to stir the peptide group solution containing PC / PMMA. About stirring conditions, what is necessary is just to employ | adopt after considering suitably conditions according to the quantity etc. of PC / PMMA added to a peptide group solution. After contacting the peptide group solution and PC / PMMA for an appropriate time, the peptide group solution and PC / PMMA are isolated by centrifugation or filtration.

[Secondary selection process]
The secondary selection step is a step of comparing peptides contained in the peptide groups before and after the contact isolation step and selecting peptides whose content in the peptide group has decreased after the contact isolation step. An outline of the contact isolation process is shown in FIG.

  The peptide group after the contact isolation step is obtained by removing the peptide specifically bound to PC / PMMA from the peptide group before the contact isolation step. Therefore, if the composition of the peptide included in the peptide group before the contact isolation process is compared with the composition of the peptide included in the peptide group after the contact isolation process, the peptide specifically bound to PC / PMMA Will decrease after the contact isolation process. And if the peptide which decreased after this contact isolation process is identified, PC / PMMA affinity peptide can be finally acquired.

  Various chromatographies including HPLC are suitable for comparison of peptide compositions in peptide groups before and after the contact isolation step. For example, when using HPLC, the HPLC chart of the sample before the contact isolation process is compared with the HPLC chart of the sample after the contact isolation process, and the reduced peak is confirmed in the sample after the contact isolation process. The peptide contained in the previous peak may be selected as a PC / PMMA affinity peptide. The decrease in the peak can be easily confirmed by comparing the peak areas. Since the peak area corresponds to the content of the peptide in the peptide group, the content of a certain peptide can be grasped by comparing the peak areas.

  Although not particularly limited, in order to select peptides that bind specifically and strongly, the peptides contained in the peptide group before and after the contact isolation step are compared, and the content in the peptide group after the contact isolation step It is preferable to select peptides having a decrease of 70% or more (more preferably 80% or more, most preferably 90% or more). In the case of using HPLC, the above-mentioned rePC / PMMA affinity peptide according to the present invention satisfies the criterion that it decreased by 70% or more after the contact isolation step.

  In addition to the above steps, the screening method of the present invention may include a step of determining the amino acid sequence of the peptide selected in the secondary selection step by mass spectrometry or the like. In addition, the screening method of the present invention may include a step of measuring the adsorption force of the peptide selected in the secondary selection step with respect to the target substance. By including the step of measuring the adsorptive power with respect to the target substance, the adsorptive power with respect to the standard substance of the finally selected peptide can be reconfirmed, and the target peptide can be screened more accurately.

  Hereinafter, examples will be shown, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Reagent used]
・ Urea (Ultra pure grade) (MP Biomedicals, Inc.)
・ CHAPS (Nacalai Tesque)
・ Dithiothreitol (DTT) (manufactured by Nacalai Tesque)
-Pharmalyte pH 3-10 (manufactured by GE Healthcare UK Ltd.)
・ Pharmalyte pH 4-6.5 (manufactured by GE Healthcare UK Ltd.)
・ Acrylamide IEF [99.9%] (Amersham Biosciences)
・ Acrylamide (manufactured by Nacalai Tesque)
・ N, N'-Methylenebisacrylamide (BIS) (Nacalai Tesque)
・ Glycerine (Wako Pure Chemical Industries, Ltd.)
・ Tris (hydroxymethyl) aminomethane (tris) (Wako Pure Chemical Industries, Ltd.)
・ 2-mercaptoethanol (Wako Pure Chemical Industries, Ltd.)
・ Butanol (Wako Pure Chemical Industries, Ltd.)
・ 6M Hydrochloric acid (HCl) (Wako Pure Chemical Industries, Ltd.)
・ Agarose (Nacalai Tesque)
・ Bromophenol blue (BPB) (Aldrich)
・ N, N, N ', N'-Tetramethylethylenediamine (TEMED) (Nacalai Tesque)
・ Ammonium Peroxodisulfate (APS) (Wako Pure Chemical Industries, Ltd.)
・ Phosphoric acid (manufactured by Nacalai Tesque)
・ Sodium dodecyl sulfate (SDS) (Pharmacia Biotech)
・ Glycine (Nacalai Tesque)
・ Methanol (Nacalai Tesque)
・ Acetic acid (Nacalai Tesque)
・ Coomassie Brilliant Blue G-250 (Nacalai Tesque)
・ Ammonium bicarbonate ≧ 99.5% (Fluka)
・ Acetonitrile [for spectral analysis] (ACN) (manufactured by Nacalai Tesque)
・ Acetonitrile [Chromasolv, for HPLC, gradient grade, ≧ 99.9%] (SIGMA-Aldrich)
・ Iodoacetamide (manufactured by GE Healthcare UK Ltd.)
・ Sequence Grade Modiffied Trypsin (trypsin) (Promega)
・ Alpha-cyano-4-hydroxy-cinnamic acid (CHCA) (manufactured by SIGMA-Aldrich)
・ Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, Ltd.)
・ Trifluoroacetic acid (for TFA high-speed liquid graph) (manufactured by Nacalai Tesque)
・ Peptide Calibration Standard II (BRUKER)
・ Sodium chloride (Nacalai Tesque)
・ Potassium chloride (Wako Pure Chemical Industries, Ltd.)
・ Di-Sodium hydrogenphosphate 12-water (Na ₂ HPO ₄ 12H ₂ O) (Nacalai Tesque)
・ Potassium dihydrogenphosphate (Nacalai Tesque)
・ Imidazole (manufactured by Nacalai Tesque)
・ Guanidine hydrochloride (Gdn-HCl) (Nacalai Tesque)
・ Bovine serum albumin (BSA) (Nacalai Tesque)
・ Sodium acetate (manufactured by SIGMA-ALDRICH)
・ BugButer Protein Extraction Reagent (BugBuster) (Novagen)
・ Benzonase Nuclease (Novagen)
・ Protease Inhibitor (Nacalai Tesque)
・ DNA Purification Kit (Promega)
・ Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd.)
・ KODplus ver.2 PCR kit (Toyobo Co., Ltd.)
・ Glycogen (manufactured by Nacalai Tesque)
・ Infusion Advantage PCR cloning kit (Clontech)
・ Chymotrypsin (Protea)
・ Micro BCA kit (Thermo)
・ DC protein assay kit (BIO-RAD)
・ Overnight Express (Novagen)
・ Ampicillin (Amp) (Nacalai Tesque)
・ 2 × YT medium (Novagen)
・ LB agar medium (Nacalai Tesque)
[Device used]
・ Ultrapure water equipment: Allium 611UV (18.2MΩcm): Sartorius
・ Electrophoresis device: Isoelectric disc electrophoresis device: Nippon Aido Co., Ltd. ・ Constant temperature double slab electrophoresis device: Nippon Aido Co., Ltd. ・ Power supply device: Electrophoresis Power Supply: GE Healthcare UK Ltd.
・ Gel photography: Image scanner (GT-X700): EPSON, Gel photography equipment (FASIII): Toyobo ・ Mass spectrometer: Voyager DE-STR: Applied Biosystems Inc
・ PH meter: Glass electrode type hydrogen ion concentration meter: HORIBA
・ Centrifuge: Table top micro cooling centrifuge 3500: KUBOTA, Universal cooling centrifuge: KUBOTA
・ Autoclave: BS-325: Tommy Seiko Co., Ltd. ・ Clean bench: NS-13B: Juji Field Co., Ltd. ・ Chromatography system: AKTAprime plus: GE Healthcare UK Ltd., His Trap HP column: GE Healthcare UK Ltd.
Dialysis membrane: Cellulose tube for dialysis (pore diameter: 400-500nm): Viskase Companies, Inc.
・ Sterilization filter: Vacuum Driven Disposable Filtration System: IWAKI
・ Ultrafiltration filter: MICROCON (registered trademark) (10,000 Nominal Molecular Weight Limit): Millipore
-HPLC pretreatment filter: Non-Sterile 4mm Millex (registered trademark) HV syringe Driven Filter Unit (450nm): Millipore
-HPLC system: PU-2089 Quaternary Gradient Pump: JASCO, LC-NetII / ADC: JASCO, MD-2018Plus Photodiode Array Detector: JASCO, UV-1575 Intelligent UV / VIS Detector: JASCO, 5C18-AR-IIpacked column (Size: 4.6 × 150mm): Nacalai Tesque Incubator: BR40LF: TAITEC
・ Thermal cycler: PCR SPRINT: Thermo
・ Spectrophotometer: UV-1600: Shimadzu Corporation ・ Freeze dryer: FD-1000: EYELA
[Used solution]
The following percentages are w / v% unless otherwise specified.
-Lysis Buffer (8M urea, 4% CHAPS, 2v / v% Pharmalyte pH 3-10, 1% DTT) ... Urea: 4.8g, 20% CHAPS: 2.0ml, Pharmalyte pH3-10: 0.20ml, DTT: 0.10 g was mixed and made up to 10 ml with ion-exchanged water.
-Anode buffer (0.068v / v% phosphoric acid): phosphoric acid: 34 μl was diluted to 50 ml with ion-exchanged water.
Cathode buffer (2v / v% TEMED) TEMED: 800 μl was diluted to 40 ml with ion-exchanged water.
-One-dimensional electrophoresis acrylamide stock solution (28.4% Acrylamide, 1.6% BIS): Acrylamide IEF: 1.42 g, BIS: 0.08 g were mixed and made up to 5 ml with ultrapure water.
・ Isoelectric gel solution (8Murea, 4% CHAPS, 2.5v / v% Pharmalyte pH 3-10,2.5v / v% Pharmalyte pH 4-6.5) (3.5% t, 5% c) ・ ・ ・ 1D electricity Acrylamide stock solution for electrophoresis: 0.29 ml, Urea: 1.2 g, 20 w / v% CHAPS: 0.5 ml, Pharmalyte pH 3-10: 62.5 μl, Pharmalyte pH 4-6.5: 62.5 μl, mix to 2.5 ml with ultrapure water Up. The isoelectric point gel solution was degassed before use. Immediately before use, 1% of 25% APS and 2.5 μl of TEMED were added as a polymerization agent.
-0.5 Mtris-HCl solution (pH 6.8) Tris: 30.3 g was adjusted to pH 6.8 with HCl, and then made up to 500 ml with ion-exchanged water.
-1Mtris-HCl solution (pH 8.8) Tris: 60.6 g was adjusted to pH 8.8 with HCl and then made up to 500 ml with ion-exchanged water.
Sample buffer for SDS-PAGE (0.0625 Mtris, 10% SDS): 0.5 Mtris-HCl solution (pH 6.8): 20 g of SDS was added to 50 ml, and the volume was made up to 400 ml with ion-exchanged water.
-SDS-PAGE electrophoresis buffer: Tris: 6 g, Glysin: 28.8 g, SDS: 2 g were dissolved in ion-exchanged water and made up to a final volume of 2000 ml.
・ Separation gel solution (0.375Mtris, 0.1% SDS, 8% glycerine) (12.3% t, 2.7% c) ・ ・ ・ 50% acrylamide: 11.64ml, 2% BIS: 6ml, 1Mtris-HCl solution (pH8.8) : 13.2 ml, 10% SDS: 0.36 ml, 60 v / v% glycerine: 4.8 ml was mixed and made up to 36 ml with ion-exchanged water. Immediately before use, 80 μl of 25% (w / v) APS and 20 μl of TEMED were added as a polymerization agent.
Stacking gel solution (0.125 Mtris, 0.1% SDS, 10% glycerine) (5.1% t, 2.6% c) ... 30% acrylamide: 1.66 ml, 2% BIS: 0.66 ml, 0.5 Mtris-HCl solution (pH 6. 8): 2.49 ml, 10% SDS: 0.1 ml, 60 v / v% glycerine: 1.66 ml were mixed and made up to 10 ml with ion-exchanged water. Immediately before use, 16 μl of 25% (w / v) APS and 10 μl of TEMED were added as a polymerization agent.
-Agarose solution for isoelectric focusing gel (0.0625Mtris, 10% SDS, 1% ager) ... SDS-PAGE sample buffer: 50 ml, Agarose: 0.5 g, BPB: trace were mixed and dissolved.
・ Fixing solution (40% methanol, 10% acetic acid): Methanol: 80 ml, Acetic acid: 20 ml were mixed and made up to 200 ml with ion-exchanged water.
-Staining solution (0.12% CBBG-250, 10% phosphoric acid, 10% ammonium sulfate, 20% methanol) ... CBB G-250: 0.24g, phosphoric acid: 20ml, ammonium sulfate: 20g, methanol: 40ml, The volume was made up to 200 ml with ion-exchanged water. Specifically, CBB G-250 and ammonium sulfate were mixed in advance, phosphoric acid was added, the volume was increased to 80% of the total amount with ion-exchanged water, and the mixture was stirred for about 10 minutes (the solution at this time was dark blue). Thereafter, 20% of the total amount of methanol was added and stirred for about 10 minutes, and used without filtration (the solution at this time was dark blue green).
-Decoloring solution (1% acetic acid) ... Acetic acid: 10 ml was made up to 1000 ml with ion-exchanged water.
-Decolorizing solution for MS sample preparation (0.1 M ammonium bicarbonate, 40 v / v% ACN) ... 1 M ammonium bicarbonate solution: 450 μl, ACN: 1.8 ml were mixed and made up to 4.5 ml with ultrapure water.
Washing solution (0.025M ammonium bicarbonate, 50% ACN): 1M ammonium bicarbonate solution: 100 μl, ACN: 2 ml were mixed and made up to 4 ml with ultrapure water.
-Digestive fluid (0.05M ammonium bicarbonate, 0.02mg / ml trypsin) ... 1M ammonium bicarbonate solution: 5µl, 0.2mg / ml trypsin: 10µl were mixed and made up to 100µl with ultrapure water. The digestion solution was prepared on ice.
Extract liquid (50% ACN, 1% TFA): ACN: 500 μl, TFA: 10 μl were mixed and made up to 1 ml with ultrapure water.
CHCA solution (saturated CHCA, 70% ACN, 0.1% TFA) CHCA: 18.9 mg, ACN: 700 μl, TFA: 1 μl were mixed and made up to 1 ml with ultrapure water.
・ 10 × PBS ... Sodium chloraide: 80 g, Potassium chloraide: 2 g, Potassium dihydrogenphosphate: 2 g, Na2HPO4 12H2O: 29 g were mixed and made up to 1000 ml with ion-exchanged water. Diluted 10 times with ion exchange water.
PBST (1 v / v% Tween 20): 10 × PBS: 100 ml, Tween 20: 10 ml were mixed and made up to 1000 ml with ion-exchanged water.
・ 2 × SDS treatment buffer (0.125Mtris, 4% SDS, 20v / v% glycerine, 2v / v% 2-mervaptoethanol) ・ ・ ・ 0.5Mtris-HCl solution (pH6.8): 0.5ml, 10% SDS: 0.8 ml, 60 v / v% glycerine: 0.67 ml, 2-mercaptoethanol: 0.04 ml, and BPB: trace were prepared.
LB agar medium: LB agar medium: 40 g was dissolved in 1000 ml of ion-exchanged water and then used after autoclaving.
2 × YT medium: 2 × YT medium: 31 g was dissolved in 1000 ml of ion-exchanged water, and then used after autoclaving.
After dissolving in 1000 ml of ion exchange water, it was autoclaved and used.
OE medium: Overnight Express: 60 g, Glycerine: 10 ml was dissolved in 1000 ml of ion exchange water, and then sterilized with a sterilization filter.
・ Solubilization buffer (6M Gdn-HCl, 10 mM 2-mercaptoethanol, 2 × PBS, pH 7.5) ・ ・ ・ Gdn-HCl: 570 g, 2-melcaptoethanol: 0.7 ml, 10 × PBS: 200 ml, mixed with ion-exchanged water The volume up to 1000ml. The pH was adjusted to pH 7.5 with NaOH.
・ Equilibration buffer for His Trap (A solution) (8M Urea, 20 mM Imidazole, 2 × PBS, pH 7.5) ・ ・ ・ Urea: 480 g, Imidazole: 1.36 g, 10 × PBS: 200 ml mixed, ion-exchanged water The volume up to 1000ml. The pH was adjusted to pH 7.5 with HCl.
・ Elution buffer for His Trap (Liquid B) (8M Urea, 400 mM Imidazole, 2 × PBS, pH 7.5) ・ ・ ・ Urea: 480 g, Imidazole: 27.2 g, 10 × PBS: 200 ml, mixed with ion-exchanged water The volume was increased to 1000 ml. The pH was adjusted to pH 7.5 with HCl.
-HPLC column equilibration buffer (liquid A) (0.1% TFA): Ultrapure water: 1000 ml and Trifluoroacetic acid (TFA high performance liquid graph): 1 ml were mixed.
-HPLC elution buffer (liquid B) (0.1% TFA, 99.9% Acetonitrile) ... Acetonitrile [Chromasolv, for HPLC, gradient grade, ≥99.9%]: 1000 ml and Trifluoroacetic acid (for TFA high performance liquid graph): 1 ml Were mixed.

[1. Screening of affinity proteins for polycarbonate or polymethyl methacrylate)
(I) Sample preparation
(1) E. coli BL21 (DE3) was inoculated from a glycerol stock into 20 ml of LB medium autoclaved in a clean bench and pre-cultured overnight at 37 ° C.
(2) The preculture was added to 100 ml of autoclaved 2 × YT medium so that OD ₆₀₀ = 0.1, and cultured at 37 ° C. and 200 rpm for 7 hours.
(3) After culturing, the culture solution was transferred to a 50 ml centrifuge tube and centrifuged at 4500 rpm for 15 minutes, and the supernatant was removed.
(4) 10 mL of BugBuster, 3 μl of Benzonase Nuclease, and 100 μl of protease inhibitor were added to the pelleted cells to lyse the cells.
(5) After centrifugation at 10000 g for 20 minutes, the supernatant was recovered and used as an adsorption sample.
(6) 10 g of a polycarbonate piece (PC piece) or polymethyl methacrylate piece (PMMA piece) (approximate surface area of about 200 cm ² ) was measured, 5 ml of the above adsorption sample was added, and the mixture was shaken at 160 rpm for 5 hours.
(7) 40 ml of PBS was added to the PC piece or PMMA piece and shaken at 80 rpm for 5 minutes. Thereafter, the supernatant was removed. This operation was performed three times.
(8) 40 ml of ultrapure water was added to the PC piece or PMMA piece and washed under the same conditions as in (7). This operation was performed three times.
(9) 4 ml of Lysis buffer was added and shaken at 160 rpm for 30 minutes, and the supernatant was recovered.
(10) 500 ml of the recovered solution was added to MICROCON (registered trademark) and centrifuged at 14000 g for 30 minutes.
(11) 100 μl of lysis buffer was added on the membrane, and after pipetting, MICROCON (registered trademark) was put in a 1.5 ml tube in the reverse direction and centrifuged at 1000 g for 3 minutes to collect the solution. This was used as an elution sample. The adsorption sample and elution sample were both stored at -20 ° C.

(II) Isoelectric focusing
(1) The inside of the glass tube was washed with ethanol. Then, a mark was placed at 11.5 cm from the bottom of the glass tube, and parafilm was wound on the bottom.
(2) 2.5 μl of APS and 1 μl of TEMED were added to the isoelectric point gel solution, and the glass tube was poured to the point where it was marked, and 20 μl of ion-exchanged water was overlaid, and incubated at room temperature until gelation.
(3) After removing the ion-exchanged water that had been overlaid, 25 μl of each sample solution was added, and 20 μl of Lysis Buffer diluted 2 times was overlaid.
(4) The buffer was filled in the anode and cathode, and isoelectric focusing was started. The voltage during electrophoresis was changed stepwise from 200 V for 1 hour, 400 V for 16 hours, and 800 V for 1 hour.
(5) The glass tube was removed from the apparatus, ion exchange water was poured between the glass tube and the gel with a syringe, and the isoelectric point gel was taken out.
(6) The isoelectric gel was transferred to a 15 ml centrifuge tube and washed 3 times with about 10 ml of ion exchange water.

(III) SDS-PAGE
(1) The glass plate was washed with ethanol, dried and assembled. Then, a mark was placed at a position 2.5 cm from the upper end of the glass plate.
(2) A solution obtained by adding 80 μl of APS and 20 μl of TEMED to the separation gel solution was poured to the position of the mark on the assembled glass plate. Thereafter, 400 ml of butanol was overlaid and incubated at room temperature until the separation gel solution polymerized.
(3) After the polymerization was completed, butanol was washed with ion-exchanged water, and water was thoroughly removed. Then, 16 μl of APS and 10 μl of TEMED were added to the stacking gel solution, and poured to the upper end of the glass plate. Thereafter, 400 μl of butanol was overlaid and incubated at room temperature until the stacking gel solution polymerized.
(4) 4.75 ml of SDS-PAGE sample buffer and 250 μl of 2-mercaptoethanol were added to the isoelectric focusing gel washed in (II) above and shaken for 15 minutes.
(5) The sample buffer for SDS-PAGE and 2-mercaptoethanol were removed, the isoelectric gel was washed with ion-exchanged water, 5 ml of sample buffer for SDS-PAGE and 0.125 g of IAA were added, and the mixture was shaken for 15 minutes.
(6) After removing the SDS-PAGE sample buffer, the isoelectric point gel was fixed on the gel prepared in (3) using the agarose solution for isoelectric point gel fixation dissolved in a microwave oven. When a molecular weight marker was added, 10 ml of the marker was soaked in a filter paper and fixed with an agarose solution for isoelectric focusing gel fixation.
(7) Electrophoresis was performed at a constant current of 20 mA for about 1 hour 30 minutes and at a constant current of 40 mA for about 3 hours. When the BPB line reached the lower end of the glass plate, the electrophoresis was terminated.
(8) The gel was removed from the glass plate and shaken in 200 ml of fixative for 2 hours or overnight.
(9) After removing the fixing solution, 200 ml of the staining solution was added and shaken for 12 hours or more.
(10) After removing the staining solution, 200 ml of decolorizing solution was added and shaken for 12 hours or more. During decolorization, the paper towel was changed about every hour for the purpose of dye adsorption.
(11) After decolorization, the image was taken with a scanner and stored at 4 ° C.

(IV) Protein identification by MALDI-TOF MS
(1) A spot to be identified was cut out from the obtained gel, and 150 μm of a decoloring solution for MS sample preparation was added and shaken for 45 minutes.
(2) The decolorizing solution was discarded, 500 μl of ultrapure water was added, and after shaking for 1 minute, the solution was discarded, and 150 μl of decoloring solution was added again and shaken for 45 minutes.
(3) 500 μl of ultrapure water was added and shaken for 1 minute. The ultrapure water was discarded and this operation was repeated twice.
(4) 200 μl of washing solution was added and shaken for 5 minutes.
(5) The washing solution was discarded, 100 μl of ACN was added and shaken for 5 minutes. Further ACN was removed and the gel was dried.
(6) 5 ml of digestive juice was added on ice, and the mixture was incubated on ice for about 20 minutes to swell.
(7) Incubate overnight (about 16 hours) at 37 ° C.
(8) The digested gel piece was taken out, 50 μl of the extract was added and shaken for 30 minutes.
(9) The extract was placed in a new 1.5 ml tube, and 25 μl of the extract was added to the gel again and shaken for 15 minutes.
(10) 25 ml of the extract was added to the above tube and frozen at -80 ° C. Then, it was made to dry by freeze-drying.
(11) 2 μl of the extract was added to the dried product, and after shaking, the solution was spun down by a centrifuge.
(12) After 0.5 μl was applied to the MS measurement plate, 0.5 μl of CHCA solution was dropped before drying.
(13) Measurement was performed using a mass spectrometer (Voyager DE-STR), and a parameter file of ACTH_reflector.bic was used.
(14) Mass spectrometry results were identified by MASCOT Peptide Mass Fingerprint. Database search conditions were database: NCBInr, Taxonomy: All entries, Enzyme: Trypsin, Fixed modifications: Carbamidomethyl, Peptide tol .: ± 0.1, Mass Values: MH +, Monoisotopic. In this example, the identifiable score was defined as 81 or more.

(result)
FIG. 3 (a) shows the two-dimensional electrophoresis result of the adsorption sample containing intracellular protein in E. coli, and FIG. 3 (b) shows the two-dimensional electrophoresis result of the eluted sample containing the protein eluted from the PC. It was shown to.

  Table 1 shows a list of proteins identified from the eluted sample. In addition, the same protein was identified also from the elution sample containing the protein eluted from PMMA by chance.

  “No.” in Table 1 corresponds to the spot number in FIG. In FIG. No. 1 spot is Malto porin (abbreviated as “MLT”), Spot No. 2 is Ompf porin (abbreviated “OMP”), No. Spot No. 3 is an elongation factor (abbreviated as “ELN”), No. The spot 4 was identified as a protein capable of specifically adsorbing bifunctional aconitate hydratase (abbreviated as “BIF”) to PC and PMMA (referred to as “PC / PMMA affinity protein”).

[2. Cloning of PC / PMMA affinity protein]
(I) Purification of E. coli genome
Extraction was performed using DNA Purification Kit (Promega).

(reagent)
Nuclei Lysis Solution
RNase Solution
Protein Precipitation Solution
DNA Rehydration Solution
(procedure)
(1) 1 ml of the culture solution of E. coli BL21 (DE3) cultured overnight was placed in a 1.5 ml tube.
(2) Centrifugation was performed at 13000 to 16000 g for 2 minutes, and the supernatant was discarded.
(3) 600 μl of Nuclei Lysis Solution was added and stirred.
(4) Incubate at 80 ° C. for 5 minutes and cool to room temperature.
(5) 3 μl of RNase Solution was added, and the mixture was stirred by inversion about 5 times.
(6) Incubate at 37 ° C. for 15-60 minutes and cool to room temperature.
(7) 200 μl of Protein Precipitation Solution was added, stirred for 20 seconds, and incubated on ice for 5 minutes.
(8) The mixture was centrifuged at 13000-16000G for 3 minutes.
(9) The supernatant containing DNA was transferred into 600 μl isopropanol at room temperature, and slowly stirred by inversion until DNA precipitated.
(10) Centrifugation was performed at 13000 to 16000 g for 2 minutes, and the supernatant was removed.
(11) 600 μl of 70% ethanol was added to wash the pellet.
(12) Centrifugation was performed at 13000 to 16000 g for 2 minutes, the supernatant was removed, and the pellet was dried.
(13) 100 μl of DNA Rehydration Solution was added and incubated at 65 ° C. for 1 hour or at 4 ° C. overnight. The extracted DNA was stored at 2-8 ° C.

(II) Isolation of PC / PMMA affinity protein gene by genomic PCR (primers used)
Primer sets for amplifying each PC / PMMA affinity protein are as follows. Two primers were used per protein.
(Primer for OMP amplification)
OMP inF S1: ATA TAC ATA TGA TGA AGC GCA ATA TTC TGG (SEQ ID NO: 23)
OMP inF S2: TAA GAA GGA GAT ATA CAT ATG AAG CGC (SEQ ID NO: 24)
OMP inF AS1: GTG CGG CCG CGA ACT GGT AAA CGA TAC CCA (SEQ ID NO: 25)
OMP inF AS2: TGG TGG TGC TCG AGT GCG GCC GCG AAC TGG (SEQ ID NO: 26)
(Primer for MLT amplification)
MLT inF S1: ATA TAC ATA TGA TGA TTA CTC TGC GCA AAC (SEQ ID NO: 27)
MLT inF S2: TAA GAA GGA GAT ATA CAT ATG ATG ATT ACT (SEQ ID NO: 28)
MLT inF AS1: GTG CGG CCG CCC ACC AGA TTT CCA TCT (SEQ ID NO: 29)
MLT inF AS2: TGG TGG TGC TCG AGT GCG GCC GCC CAC CAG (SEQ ID NO: 30)
(ELN amplification primer)
ELN inF S1: ATA TAC ATA TGT CTA AAG AAA AGT TTG A (SEQ ID NO: 31)
ELN inF S2: TAA GAA GGA GAT ATA CAT ATG TCT AAA GAA (SEQ ID NO: 32)
ELN inF AS1: GTG CGG CCG CGC TCA GAA CTT TTG CTA (SEQ ID NO: 33)
ELN inF AS2: TGG TGG TGC TCG AGT GCG GCC GCG CTC AGA (SEQ ID NO: 34)
(Primer for BIF amplification)
BIF inF S1: ATA TAC ATA TGG TGC TAG AAG AAT ACC GTA (SEQ ID NO: 35)
BIF inF S2: TAA GAA GGA GAT ATA CAT ATG GTG CTA GAA (SEQ ID NO: 36)
BIF inF AS1: GTG CGG CCG CAA CCG CAG TCT GGA AAA TCA (SEQ ID NO: 37)
BIF inF AS2: TGG TGG TGC TCG AGT GCG GCC GCA ACC GCA (SEQ ID NO: 38)
(procedure)
(1) Each of the above primers was diluted to 100 nmol / ml.
(2) 180 μl of distilled water and 20 μl of the diluted solution prepared in (1) were mixed to obtain a 10 nmol / ml primer solution.
(3) 33 μl of distilled water in a PCR tube, 5 μl of KODbuffer, 5 μl of dNTP, 2 μl of MgSO ₄ , 1 μl of E. coli genomic DNA extracted by (I), 1.5 μl of each of the two primer solutions prepared in (2) Mixed one by one.
(4) After centrifugation, amplification was performed with a thermal cycler. The thermal cycler was run with the following program.
Pre Denature: 94 ° C for 2 min, Denature: 94 ° C for 15 sec, Annealing: (Tm-5) ° C for 30 sec, Extension: 68 ° C for 1 min or 3 min, and 30 cycles from Denature to Extension.
(III) Construction of gene expression vector for PC / PMMA affinity protein
In-Fusion ^™ Advantage PCR Cloning Kit (Clontech) was used.

(reagent)
In-Fusion Enzyme
5 × In-Fusion Reaction Buffer
Cloning Enhancer
(procedure)
(1) The pET22 (b) Vector was digested with restriction enzymes (NdeI, NotI).
(2) 5 μl of the PCR reaction solution prepared in (II) and 2 μl of Cloning Enancer were mixed, incubated at 37 ° C. for 15 minutes, and then incubated at 80 ° C. for 15 minutes.
(3) 3.5 μl of pET22 (b) Vector cleaved with a restriction enzyme, 2 μl of 5 × In-Fusion Reaction Buffer, and 1 μl of In-Fusion Enzyme were mixed and incubated at 37 ° C. for 15 minutes and at 50 ° C. for 15 minutes.
(4) 40 μl of distilled water, 5 μl of sodium acetate, 1 μl of glycogen, and 135 μl of ethanol were added and stirred with a vortex mixer.
(5) Centrifugation was performed at 20000 g for 2 minutes, and the supernatant was removed.
(6) 500 μl of ice-cold 70% ethanol was added, and after stirring, centrifuged at 20000 g for 2 minutes to remove the supernatant.
(7) 3 μl of distilled water was added to the dried pellet and dissolved.
(8) The solution prepared in (7) was added to the competent cell, incubated at 42 ° C. for 45 seconds, plated on an LB Ager plate and incubated overnight at 37 ° C.

(IV) Expression of PC / PMMA affinity protein
(1) Autoclaved in a clean bench and added to 20 ml of 2 × YT medium so that the concentration of Amp was 50 μg / ml, and transformed E. coli BL21 (DE3) was inoculated and pre-cultured overnight at 37 ° C.
(2) The preculture was added to 100 ml of OE medium to which Amp was added in the same manner as in (1) so that OD ₆₀₀ = 0.1, and cultured at 37 ° C. and 200 rpm for 24 hours.
(3) After the culture, the culture solution was transferred to a 50 ml centrifuge tube and centrifuged at 4500 rpm for 15 minutes, and the supernatant was removed.
(4) Solubilization buffer (6M Gdn-HCl, 10 mM 2-mercaptoethanol, 2 × PBS, pH 7.5) was added to the pellet and dissolved.

(V) Purification by affinity chromatography
(1) Liquid A (8M Urea, 20 mM Imidazole, 2 × PBS, pH 7.5) and liquid B (8M Urea, 400 mM Imidazole, 2 × PBS, pH 7.5) were prepared.
(2) A ^* KTA system was started, and A liquid and B liquid were set to Line A and B (however, A ^{* on the} left indicates A umlaut).
(3) Lines A and B were replaced with liquids A and B, and a His Trap ^™ HP column was attached.
(4) Solution A was supplied to the column at a flow rate of 1 ml / min to equilibrate the interior of the column.
(5) Each protein solution was supplied at a flow rate of 1 ml / min and adsorbed onto the column.
(6) Liquid A was supplied to the column at a flow rate of 1 ml / min and washed.
(7) Liquid B was supplied to the column at a flow rate of 1 ml / min, and the target protein was recovered with a fraction collector.
(8) The collected target protein solution was dialyzed against 8M Urea + PBS.

(VI) Confirmation of purification by SDS-PAGE
(1) The glass plate was washed with ethanol, dried and assembled. Then, a mark was placed at a position 2.5 cm from the upper end of the glass plate.
(2) 80 μl of APS and 20 μl of TEMED were added to the separation gel solution, and poured to the marked position on the assembled glass plate. Thereafter, 400 μl of butanol was overlaid and incubated at room temperature until the separation gel solution polymerized.
(3) After polymerization, butanol was washed with ion-exchanged water, carefully drained, APS 16 μl and TEMED 10 μl were added to the stacking gel solution, and poured to the upper end of the glass plate. Thereafter, comb was applied, 400 μl of butanol was overlaid, and incubated at room temperature until the stacking gel solution was polymerized.
(4) 20 μl of the sample solution and 20 ml of 2 × SDS processing buffer were mixed and incubated at 90 ° C. for 5 minutes.
(5) 30 μl of sample was added and electrophoresis was started.
(6) Electrophoresis was performed at a constant current of 20 mA for about 1 hour 30 minutes and at a constant current of 40 mA for about 3 hours. The electrophoresis was terminated when the BPB line reached the lower end.
(7) The gel was removed from the glass plate and shaken in 200 ml of fixative solution for 2 hours or overnight.
(8) After removing the fixing solution, 200 ml of the staining solution was added and shaken for 12 hours or more.
(9) After removing the staining solution, 200 ml of decolorizing solution was added and shaken for 12 hours or more. During decolorization, the paper towel was changed about every hour for the purpose of dye adsorption.
(10) After decolorization, the image was taken with a scanner and stored at 4 ° C.

(VII) Protein quantification For protein quantification, a detection kit (DC Protein Assay (manufactured by BIO-RAD)) based on the method of Lowry et al. Was used.

(reagent)
Reagent A
Reagent B
Reagent C
Reagent A '... Reagent A: Reagent S = 50: 1 mixed solution (Procedure)
(1) The standard solution (BSA) was diluted with 8M Urea + PBS so that the concentration was 0, 125, 250, 500, 1000 μg / ml, and 100 μl was prepared respectively.
(2) Each purified protein after dialysis was diluted 5 times or 25 times with 8M Urea + PBS so as to be 100 μl.
(3) 500 μl of Reagent A ′ was added to the above solutions (1) and (2) and stirred. Thereafter, 4 ml of Reagent B was added and incubated at room temperature for 15 minutes.
(4) The absorbance at a wavelength of 750 nm was measured.
(5) A calibration curve was created from the measurement results of the standard solution, and the concentration of each purified protein was calculated.

(result)
When the gene of PC / PMMA affinity protein was cloned from the E. coli genome by PCR and overexpressed in E. coli BL21 (DE3), it was recovered as an inclusion body. These were solubilized with Solubilization buffer and purified in a denatured state using a His Trap HP column. After purification, it was confirmed by SDS-PAGE that the protein was expressed and purified. The result is shown in FIG.

[3. PC / PMMA affinity peptide screening]
(I) Micro BCA assay For the micro BCA assay, Micro BCATM Protein Assay Kit (manufactured by Therom) was used.

(reagent)
Reagent MA
Reagent MB
Reagent MC
Albumin Standard
(procedure)
(1) Each purified protein was diluted with PBS to 25 mg / ml.
(2) 4 g of PC piece or PMMA piece (approximate surface area of about 80 cm ² ) was measured, 2 ml of each diluted protein solution was added, and the mixture was shaken at 25 ° C. and 200 rpm for 2 hours.
(3) The PC piece or PMMA piece was washed 5 times with 25 ml of PBS.
(4) The standard solution (Albumin Standard) was adjusted to 40, 20, 10, 5, 2.5, 1.25, 0.625 μg / ml.
(5) 1 ml of PBS was added to the PC piece or PMMA piece washed in (3) above.
(6) The mixture was mixed so that the volume ratio was Reagent MA: Reagent MB: Reagent MC = 25: 24: 1, and 1 ml was added to each sample prepared in the above (4) and (5). After stirring, it was incubated at 37 ° C. for 2 hours.
(7) Absorbance at a wavelength of 562 nm was measured.
(8) A calibration curve was created from the measurement results of the standard solution, and the adsorption amount was calculated.

(result)
Four types of purified proteins and bovine serum albumin (BSA) as a control were diluted to 25 μg / ml in PBS, and the amount adsorbed on PC pieces or PMMA pieces was measured. FIG. 5 shows the result. As shown in FIG. 5, the four types of proteins screened showed higher amounts of adsorption than the control BSA, indicating that they had an affinity for PC pieces or PMMA pieces. In particular, OMP and MLT showed a high adsorption amount.

  In the same experiment using PBST containing 1% of nonionic surfactant, the amount of adsorption was remarkably reduced. From these results, it was suggested that these PC / PMMA affinity proteins were adsorbed mainly by hydrophobic interaction.

(II) Protein digestion adsorption experiment (Procedure)
(1) Each purified protein was diluted with PBS to 500 μg / ml.
(2) 20 μg of trypsin or chymotrypsin was added to 1 ml of the diluted solution, and the mixture was incubated at 37 ° C. for 12 hours or longer for digestion.
(3) 600 μl of the digested solution was added to 16 g of PC piece or PMMA piece (approximate surface area of about 330 cm ² ) and shaken at 25 ° C. and 200 rpm for 2 hours.
(4) Liquid A (0.1% TFA) and liquid B (0.1% TFA, 99.9% ACN) were prepared.
(5) After starting the HPLC system, A liquid and B liquid were set in Line A and B, and Line A and B were replaced with A liquid and B liquid.
(6) Solution A was supplied to the column at a flow rate of 1 ml / min to equilibrate the column.
(7) The liquid diluted in (2) above and the liquid adsorbed after digestion were filtered through a pretreatment filter and then supplied to a 100 ml column.
(8) The concentration of solution B was increased linearly, and the peptide was eluted from the column.
(9) Compared before and after adsorption, an eluate corresponding to a peak in which a significant decrease was observed was collected.

(result)
The result of having compared the peptide component before and behind adsorption | suction to PC piece or PMMA piece by HPLC is shown in FIGS.

  FIG. 6 shows an HPLC chart of a tryptic digest of BSA as a control. The “before adsorption” chart in FIG. 6 is a chart before the BSA trypsin digest is adsorbed to the PC piece, and the “after adsorption” chart is a chart after the BSA trypsin digest is adsorbed to the PC piece. “Acetonitril%” indicates the concentration of acetonitrile in the eluate.

  Regarding the tryptic digest of BSA, no significant decrease in the peak after adsorption of the PC piece was observed. The data were omitted, but the results for the chymotrypsin digest of BSA were similar. In addition, although the data is omitted, there was no particular decrease in the peak after the adsorption of the PMMA piece. That is, it can be said that there is no peptide that can be specifically and strongly adsorbed to PC or PMMA in the BSA tryptic digest and chymotrypsin digest. When the peak area decreased by 70% or more in the sample after adsorption, it was determined that “the peak was significantly decreased” (the same applies hereinafter).

  Fig. 7 (a) shows an HPLC chart of a BIF tryptic digest, and Fig. 7 (b) shows an HPLC chart of a BIF chymotrypsin digest. The “Before Adsorption” chart in FIG. 7 is a chart before the BIF enzyme digest is adsorbed to the PC piece, and the “After Adsorption” chart is a chart after the BIF enzyme digest is adsorbed to the PC piece. “Acetonitril%” indicates the concentration of acetonitrile in the eluate.

  Regarding the BIF tryptic digest and chymotrypsin digest, there was no particular decrease in the peak after adsorption of the PC piece. In addition, although the data is omitted, there was no particular decrease in the peak after the adsorption of the PMMA piece. That is, it can be said that there is no peptide that can specifically and firmly adsorb to PC or PMMA in the BIF tryptic digest and chymotrypsin digest.

  FIG. 8A shows an HPLC chart of the digested product of MLT trypsin, and FIG. 8B shows an HPLC chart of the digested product of MLT chymotrypsin. The “before adsorption” chart in FIG. 8 is a chart before the enzyme digest of MLT is adsorbed to the PC piece, and the “after adsorption” chart is a chart after the enzyme digest of MLT is adsorbed to the PC piece. “Acetonitril%” indicates the concentration of acetonitrile in the eluate.

  About the tryptic digest of MLT, the peak which has decreased remarkably after adsorption | suction of PC piece was confirmed (peaks 1-3 in FIG. 8 (a)). It can be said that the peptides contained in these three peaks are peptides that can be adsorbed specifically and firmly to PC. For the chymotrypsin digest of MLT, several peaks that were significantly reduced after adsorption of the PC pieces were confirmed. However, mass spectrometry by MALDI-TOF MS was performed, and the amino acid sequence was searched using a database. As a result, the peptide sequence could not be identified. In addition, although the data is omitted, there was no particular decrease in the peak after the adsorption of the PMMA piece.

  FIG. 9A shows an HPLC chart of the OMP tryptic digest, and FIG. 9B shows an HPLC chart of the OMP chymotrypsin digest. The “before adsorption” chart in FIG. 9 is a chart before the enzyme digest of OMP is adsorbed to the PC piece, and the “after adsorption” chart is a chart after the enzyme digest of OMP is adsorbed to the PC piece. “Acetonitril%” indicates the concentration of acetonitrile in the eluate.

  For the tryptic digest of OMP, three peaks that were significantly reduced after adsorption of the PC pieces were confirmed (peaks 4 to 6 in FIG. 9A). It can be said that the peptides contained in these three peaks are peptides that can be adsorbed specifically and firmly to PC. In addition, although the data is omitted, three peaks that are markedly reduced after adsorption of the PMMA pieces were confirmed for the tryptic digest of OMP.

  FIG. 10 (a) shows an HPLC chart of an ELN tryptic digest, and FIG. 10 (b) shows an HPLC chart of an ELN chymotrypsin digest. The “before adsorption” chart in FIG. 10 is a chart before the enzyme digest of ELN is adsorbed to the PC piece, and the “after adsorption” chart is a chart after the enzyme digest of ELN is adsorbed to the PC piece. “Acetonitril%” indicates the concentration of acetonitrile in the eluate.

  Regarding the tryptic digest of ELN, one peak that significantly decreased after adsorption of the PC piece was confirmed (peak 7 in FIG. 10 (a)). The peptide contained in one peak can be said to be a peptide that can be adsorbed specifically and firmly to PC. In addition, although the data is omitted, there was no particular decrease in the peak after the adsorption of the PMMA piece.

  In addition, when digestion was performed with chymotrypsin that specifically cleaves the C-terminal side of aromatic amino acids, almost no decrease in peak was observed with proteins other than MLT. From this result, it was suggested that aromatic amino acids are involved in adsorption to PC.

(III) Determination of amino acid sequence by MALDI-TOF MS
(1) The eluate corresponding to the peak (peaks 1 to 7) collected in the above (II) was dried by lyophilization.
(2) 2 μl of the extract was added to the dried product, and the mixture was shaken and spun down.
(3) After 0.5 μl was applied to the MS measurement plate, 0.5 μl of CHCA solution was dropped before drying.
(4) The analysis was performed using a mass spectrometer (Voyager DE-STR). A parameter file of ACTH_reflector.bic was used for mass spectrometry.
(5) The molecular weight result obtained by mass spectrometry was compared with the database (ExPASy Proteomics server: http://www.expasy.ch/) to determine the amino acid sequence.

(result)
The amino acid sequences of the determined peaks 1 to 7 are shown in Tables 2 to 5. “Peak No.” in Tables 2 to 5 corresponds to the number of the peak found in (II) above, “MW” is the molecular weight of the peptide, and “position” is the peptide amino acid sequence in the amino acid sequence before enzymatic digestion. “(Secondary structure)” indicates the secondary structure of the peptide in the protein before enzymatic digestion. The symbol “a” indicating the secondary structure indicates an α-helix structure, “b” indicates a β-sheet structure, “c” indicates a coil, and “t” indicates a turn.

  Table 2 shows the amino acid sequences of peaks 1 to 3 and the like that significantly decreased after the adsorption of the PC piece for the tryptic digest of MLT. Peaks 1 and 2 were the same peptide.

  Table 3 shows the amino acid sequences of peaks 4 to 6 and the like that were significantly reduced after adsorption of the PC pieces for the tryptic digest of OMP.

  Table 4 shows the amino acid sequence of peak 7 and the like that significantly decreased after adsorption of the PC piece for the tryptic digest of ELN.

  Table 5 shows the amino acid sequences and the like of the three peaks markedly reduced after the adsorption of the PMMA pieces for the tryptic digest of OMP. PMOMP21 and PMOMP3 in Table 3 were the same peptide. That is, it was found that PMOMP21 (PMOMP3) is a peptide that can be adsorbed specifically and firmly to PC and PMMA.

[4. Measurement of affinity peptide adsorption force)
(procedure)
(1) 100, 75, 50, 25, 12.5, 6.25 μg / ml peptide solutions were prepared. PCOMP3 (PMOMP21) is dissolved in 2% DMSO + PBS, PCOMP6 is dissolved in 2% DMSO + PBS, PCOMP7 is dissolved in 1% DMSO + PBS, PCMLT8 is dissolved in 1% DMSO + PBS, PCMLT10 Is dissolved in 0.8M urea + PBS, PCELN8 is dissolved in 0.08M urea + PBS, PMOMP19 is dissolved in 0.8M urea + PBS 0.8M urea, PMOMP25 is dissolved in 0.8M urea + PBS, Standard1 and Standard2 are Dissolved in PBS. Standard 1 (GERGFFYTPKA: SEQ ID NO: 39) containing a large amount of β-sheet structure and Standard 2 (NPKYEQFLE: SEQ ID NO: 40) containing a large α-helix structure were used as comparative peptides.
(2) 400 μl of the prepared solution was added to 3 g of PC piece or PMMA piece (approximate surface area of about 60 cm ² ) and shaken at 25 ° C. and 200 rpm for 2 hours.
(3) Liquid A (0.1% TFA) and liquid B (0.1% TFA, 99.9% ACN) were prepared.
(4) After the HPLC system was started, liquid A and liquid B were set in Line A and B, respectively, and Line A and B were replaced with liquid A and liquid B, respectively.
(5) Solution A was supplied to the column at a flow rate of 1 ml / min to equilibrate the interior of the column.
(6) The liquid before and after adsorption of the PC piece or PMMA piece was applied to the column in an amount of 100 μl.
(7) The concentration of solution B was increased linearly, and the peptide was eluted from the column.
(8) A calibration curve was drawn with the sample before adsorption, and an adsorption isotherm was created from the peak height after adsorption.

(result)
FIG. 11 shows the results of measuring the adsorption force of each peptide on PC. In Fig. 11, the black diamond symbol is Standard1, the white diamond symbol is Standard2, the black circle symbol is PCOMP3, the white circle symbol is PCOMP7, the black triangle symbol is PCMLT8, and the white triangle symbol is PCELN8. Show. The results for PCMLT10 and PCOMP6 are omitted.

  FIG. 12 shows the results of measuring the adsorption power of each peptide to PMMA. In FIG. 12, the black diamond symbol indicates the result of Standard1, the open diamond symbol indicates Standard2, the black circle symbol indicates PMOMP21, the open circle symbol indicates PMOMP19, and the black triangle symbol indicates the result of PMOMP25.

10 and 11, the horizontal axis indicates the concentration of unadsorbed peptide (μM), and the vertical axis indicates the adsorption density (μmol / m ² ). 10 and 11, the steep slope of the curve with respect to the horizontal axis (unadsorbed peptide concentration) means that the adsorptive power is higher.

  According to FIGS. 10 and 11, any peptide identified as an affinity peptide showed very high adsorption power to PC or PMMA compared to the comparative peptide. In particular, PCOMP3 (PMOMP21) was able to adsorb to both PC and PMMA, and because all unadsorbed peptides were not detected at the peptide concentration examined this time, it was found that the adsorption power was extremely high. .

According to the calculation by the inventors, the adsorption density of the peptide specifically binding to PC or PMMA shown in Patent Document 2 and Non-Patent Document 1 is about 1.0 μmol / m ^{2 at the} maximum. In addition, it can be said that each peptide has an extremely high adsorption power for PC and / or PMMA.

  In addition, although the two-dimensional structure of the peptide discovered this time as an affinity peptide was put together in Tables 2-5, many (beta) -sheet structures were contained in the peptide with strong adsorption power. Therefore, it was suggested that the β-sheet structure is involved in the adsorption of the affinity peptide to PC or PMMA.

  The peptide according to the present invention is capable of specifically and firmly binding to polycarbonate and / or polymethyl methacrylate. Therefore, by introducing the peptide into a biomolecule such as a protein, a desired protein can be immobilized in a high density and orientation on at least one of a polycarbonate substrate and a polymethyl methacrylate substrate. Since the introduction position of the peptide according to the present invention can be arbitrarily controlled, it can be expected that the bioactivity of the biomolecule can be maintained. Therefore, the peptide according to the present invention can greatly contribute to the development of materials in the biochip and life science fields.

  Moreover, according to the screening method of the present invention, peptides capable of binding to various target substances such as polycarbonate and polymethyl methacrylate can be efficiently screened. Therefore, the screening method of the present invention can greatly contribute to immobilization of biomolecules such as proteins.

Claims (14)

A ligand peptide used to specifically bind at least one of polycarbonate and polymethyl methacrylate by introducing the protein into a desired protein, which is shown in (a) or (b) below. And a peptide having a molecular weight of 10 kD or less:
(A) a peptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8;
(B) in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and polycarbonate and polymethyl methacrylate A peptide having an activity of specifically binding to at least one of the above. A ligand peptide used to specifically bind the protein to both polycarbonate and polymethyl methacrylate by introduction into the desired protein, the peptide shown in (c) or (d) below: A ligand peptide comprising: and having a molecular weight of 10 kD or less:
(C) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4;
(D) The amino acid sequence shown in SEQ ID NO: 4 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and specifically binds to polycarbonate and polymethyl methacrylate A peptide having the activity of   The ligand peptide according to claim 1 or 2, wherein the molecular weight is 3.5 kD or less. A method for immobilizing a protein, wherein a desired protein is specifically bound to and immobilized on at least one of polycarbonate and polymethyl methacrylate,
An introduction step of introducing the ligand peptide according to any one of claims 1 to 3 into the protein, and a protein into which the ligand peptide is introduced obtained by the introduction step, and at least polycarbonate and polymethyl methacrylate A method for immobilizing a protein comprising a contact step of bringing one or more into contact with each other.   The method for immobilizing a protein according to claim 4, wherein the introducing step is a step of producing a fusion protein of the desired protein and the ligand peptide according to any one of claims 1 to 3. A method for producing a protein that specifically binds to at least one of polycarbonate and polymethyl methacrylate,
A method for producing a protein, comprising an introduction step of introducing the ligand peptide according to any one of claims 1 to 3 into the protein.   The method for producing a protein according to claim 6, wherein the introducing step is a step of producing a fusion protein of the desired protein and the peptide according to any one of claims 1 to 3.   A protein complex formed by immobilizing a protein produced by the method for producing a protein according to claim 6 or 7 on a base material made of polycarbonate or polymethyl methacrylate.   A protein chip, wherein the protein produced by the protein production method according to claim 6 or 7 is immobilized on a substrate made of polycarbonate or polymethyl methacrylate. A method for screening a peptide that specifically binds to a target substance,
A primary selection step of selecting a protein that specifically binds to the target substance by contacting the protein library with the target substance;
A peptideization step of preparing a peptide group by fragmenting the protein selected by the primary selection step;
After contacting the peptide group prepared in the peptidization step with the target substance, the contact isolation process for isolating the peptide group and the target substance, and the peptides contained in the peptide group before and after the contact isolation process are compared, A secondary selection step of selecting peptides whose content in the peptide group has decreased after the contact isolation step,
A screening method comprising the steps of:   The secondary selection step is a step of comparing peptides contained in peptide groups before and after the contact isolation step and selecting peptides whose content in the peptide group is reduced by 70% or more after the contact isolation step. Item 11. The screening method according to Item 10.   The screening method according to claim 10 or 11, wherein the target substance is an organic polymer.   The screening method according to any one of claims 10 to 12, wherein the target substance is one or more selected from the group consisting of polycarbonate and polymethyl methacrylate.   The screening method according to any one of claims 10 to 13, wherein the protein library comprises intracellular proteins.