JP2020007309A - Method for producing chemically cross-linked peptide, chemically cross-linked peptide prepared using the method, and peptide library by cdna display method using the constructed peptide - Google Patents

Abstract

To provide a chemically cross-linked peptide having a stable structure under a reducing condition.SOLUTION: An mRNA is prepared by transcription from a DNA having a given sequence. The mRNA is subjected to ligation to an mRNA binding site of a linker having a peptide binding site, a solid phase binding site and an mRNA binding site for preparing a linker. The linker is translated in a cell-free translation system so as to synthesize a peptide corresponding to the mRNA in a state where it is bound to the peptide binding site of the linker. The peptide-bound linker is immobilized on a solid phase. An S-S bond in the peptide bound to the linker which has been immobilized on the solid phase is cleaved by reduction. Then, a divalent cross-link forming compound so selected to form a cross-link is bound to the reduced -SH group so as to form an intramolecular cross-link in a peptide. The chemically cross-linked peptide has 90% or more of cross-linking efficiency to the linker in a gel shift assay.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a chemically crosslinked peptide, the peptide produced using the method, and a peptide library. In particular, the present invention relates to a method for producing a chemically crosslinked peptide which does not lose its function even under reducing conditions, a chemically crosslinked peptide produced by using the method, and a peptide library prepared by using the crosslinked peptide by cDNA display method.

  In order to properly diagnose various diseases and to carry out effective treatment, it is very important to detect proteins and other biomolecules serving as disease markers. Conventionally, for detection of such a biomolecule, an antibody molecule (hereinafter, sometimes simply referred to as “antibody”) that can specifically bind to a biomolecule to be detected (hereinafter, referred to as “target molecule”) is used. Thus, methods for detecting based on the interaction between these two molecules have been widely used.

  By the way, antibodies, such as immunoglobulin G, which have been used by those skilled in the art for testing as described above, have a large molecular weight and a complicated structure, as is apparent from their structures. For this reason, there has been a problem that chemical synthesis cannot be performed and specific modification is difficult. Further, there is a problem that the thermal stability is poor.

  To compensate for these drawbacks, peptide molecules have come to the spotlight. These peptide molecules have a smaller molecular weight than the above-mentioned antibodies, so that they can be chemically synthesized and can be specifically modified. Furthermore, there is an advantage that the thermostability is higher than that of an antibody.

  By the way, as a peptide having a naturally occurring intramolecular crosslink, conotoxin can be exemplified. Conotoxins are shell venoms (neurotoxins) made from a mixture of various peptides produced by the mussel, and have three disulfide bridges (see Non-Patent Document 1).

  Peptides have been improved for the purpose of stabilizing the peptide structure, and Cys4-2 or Cys2-6 having an intramolecular crosslink (a synthetic IL-6 receptor having an intramolecular crosslink (hereinafter, “IL-6R”). ), IL-6R is a receptor for interleukin 6, which is an inflammatory cytokine, and the like (see Non-Patent Document 2, hereinafter referred to as "prior art 1"). A peptide having such an intramolecular crosslink has an advantage that its structure is stable as compared with a peptide having no intramolecular crosslink.

PDB: 1TTK <http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl> Yamaguchi, et al., Nucleic Acid Research, 37, e108 (2009)

  Prior art 1 is an excellent technique in that the structure is stabilized by forming a disulfide bridge in the peptide. However, when such an intramolecularly crosslinked peptide is used during a reaction under reducing conditions, the disulfide bridge is cleaved, and thus there is a problem that a peptide having such a disulfide bridge cannot be used under reducing conditions. Then, when the disulfide bridge is cleaved, the structure of the peptide becomes unstable, and if the intramolecular bridge is maintained, the function that the peptide should originally exert cannot be exhibited, There was a very big problem.

In order to specifically modify a peptide, it is necessary to react under various conditions. However, when reducing conditions cannot be used, there is a problem that only very limited modification can be performed. Occurs.
For this reason, there is a very strong social demand for maintaining intramolecular crosslinks in peptide molecules even under reducing conditions.

Under these circumstances, the inventors of the present invention have made extensive studies and completed the present invention.
That is, the first aspect of the present invention is a method for producing a chemically crosslinked peptide having a stable crosslinked structure even under reducing conditions, comprising: (a) an mRNA preparation step of preparing mRNA by transcription from DNA having a desired sequence; (B) a linker preparation step of ligating the mRNA obtained in the step (a) to an mRNA binding site of a linker having a peptide binding site, a solid phase binding site and an mRNA binding site; and (c) the (b) (D) translating the linker obtained in the step with a cell-free translation system and synthesizing the peptide corresponding to the mRNA in a state bound to the peptide binding site of the linker; Immobilizing the obtained linker on a solid phase; and (e) reducing the SS bond in the peptide bonded to the linker immobilized on the solid phase in step (d). Cut by A chemical cross-linking step of forming an intramolecular cross-linking of the peptide by binding the compound for cross-linking to form an —SH group and then forming a cross-link, and the reduced —SH group. The compound for use is a divalent cross-linking agent selected from the group consisting of bismaleimide ethane, 1,6-bismaleimide hexane, 1,4-bismaleimide butane, and 1,4-bismaleimidyl-2,3-dihydroxybutane. A method for producing a chemically crosslinked peptide, characterized in that:

  Here, it is preferable that the peptide binding site is composed of any one selected from the group consisting of puromycin, AANS-amino acid, and PANS-amino acid. Further, it is preferable that the solid phase binding site is constituted by one selected from the group consisting of streptavidin and biotin. Further, the crosslinking compound is preferably a divalent crosslinking agent, such as bismaleimide ethane, 1,6-bismaleimide hexane, 1,4-bismaleimide butane, 1,4-bismaleimidyl-2,3-dihydroxy. It is preferably selected from the group consisting of butane, 1,8-bis-maleimide diethylene glycol, 1,11-bis-maleimide triethylene glycol, and other analogs of bismaleimide ethane.

  A second aspect of the present invention is a chemically crosslinked peptide having a crosslinked structure that is stable even under reducing conditions produced by the above method for producing a chemically crosslinked peptide. Here, the chemically cross-linked peptide preferably has at least one cross-linking site, and the cross-linked peptide preferably has an outer diameter of 60 nm to 100 nm.

  A third aspect of the present invention is a method for purifying an interacting protein, wherein a protein that interacts with these peptides is purified using a chemically crosslinked peptide having a crosslinked structure that is stable even under the above reducing conditions.

  A fourth aspect of the present invention is a method for screening interacting proteins, wherein a protein that interacts with these peptides is screened using chemically crosslinked peptides having a crosslinked structure that is stable even under the above reducing conditions.

  A fifth aspect of the present invention is a peptide library by a cDNA display method, which comprises a chemically crosslinked peptide having a crosslinked structure that is stable even under the above reducing conditions.

  According to the method for producing a chemically crosslinked peptide of the present invention, a method for producing a chemically crosslinked peptide having a stable crosslinked structure even under reducing conditions is provided. Furthermore, in the chemically crosslinked peptide of the present invention, a peptide that forms a disulfide bridge (hereinafter, referred to as “naturally crosslinked peptide”) interacts with the protein (hereinafter, referred to as “interacting protein”). Can provide a chemically crosslinked peptide capable of interacting with a natural crosslinked peptide. Furthermore, by using the chemically crosslinked peptide of the present invention, the above-mentioned interacting proteins can be screened and produced. In addition, it is possible to provide a high-quality peptide library composed of only a peptide having an intramolecular cross-link formed by a cDNA library method.

FIG. 1 is a schematic diagram showing an mRNA-linker conjugate in which mRNA is linked to a puromycin linker used in the present invention. FIG. 2a is a schematic view showing a procedure (steps 1 to 8) for preparing a solid phase conjugate in which an mRNA-pulldown linker-peptide conjugate to which a chemically cross-linked peptide having a desired sequence is bonded is bonded. FIG. 2B is an enlarged schematic view showing the reactions of Steps 7 and 8 in FIG. 2A. FIG. 3 is a schematic diagram showing the procedure (steps 8 to 11) of how to separate uncrosslinked peptides after chemical crosslinking, and the products. FIG. 3a is a schematic diagram showing the state of the solid phase conjugate after the chemical crosslinking. FIG. 3b is a schematic diagram showing the product after reduction and biotinylation treatment after chemical crosslinking. FIG. 3c is a schematic diagram showing a state after the obtained product is subjected to an enzyme treatment. FIG. 3d is a schematic diagram showing the state after the obtained product has been treated with neutravidin. FIG. 4 shows the results of experiments confirming the effect of the reduction treatment on chemical crosslinking. (A) is a photograph showing the results of gel electrophoresis (SDS-PAGE) after reduction treatment applied to the uncrosslinked peptide and the crosslinked peptide after reduction treatment. (B) is a graph when (A) is quantified. FIG. 5 shows the results when a solid phase conjugate to which a peptide (Cys2-6) interacting with IL-6R is immobilized or a solid phase conjugate to which a peptide not interacting (BDA, FLAG) is immobilized is used. FIG. 4 is a diagram schematically showing a reaction product of Example 1 and results of SDS-PAGE. FIG. 5a is a diagram schematically showing a reaction product when Cys2-6 is used and that the finally obtained protein is IL-6R. FIG. 5b is a diagram schematically showing a reaction product when BDA or FLAG is used, and that the finally obtained protein is not IL-6R. FIG. 5c is a diagram schematically showing the result of SDS-PAGE of each product obtained above. FIG. 6 is a photograph showing the results when a positive control (IL-6R), a negative control, a Cys2-6 peptide having a different cross-linking state, BDA and FLAG were subjected to SDS-PAGE.

Hereinafter, the present invention will be described in more detail with reference to FIGS.
As shown in FIG. 2A, the present invention provides (a) an mRNA preparation step of preparing mRNA from a DNA having a desired sequence by transcription; and (b) converting the mRNA obtained in the (a) step into a peptide binding site, A linker preparation step of ligating to an mRNA binding site of a linker having a solid phase binding site and an mRNA binding site; and (c) translating the linker obtained in the step (b) by a cell-free translation system to correspond to the mRNA. (D) a peptide synthesis step of synthesizing a peptide to be bound to the peptide binding site of the linker; (d) an immobilization step of immobilizing the linker obtained in the step (c) on a solid phase; ) The SS bond in the peptide bound to the linker immobilized on the solid phase in the step (d) is cleaved by reduction to a -SH group, and then a crosslink having a width of 60 nm to 80 nm is formed. Crosslinking compound and By combining the reduced -SH groups, chemical cross-linking step of forming intramolecular cross-linking of the peptide; characterized in that it comprises a a method for manufacturing a chemical crosslinking peptide.

(1) Chemically Cross-Linked Peptide In the present specification, a “chemically cross-linked peptide” is a nucleic acid molecule that specifically binds to a specific substance and refers to a substance that changes the function of a protein or cell. "Specific substances" to which the peptide aptamer binds include growth factors, enzymes, receptors, viral proteins and other various proteins, various metal ions, and the like. Peptide aptamers can be synthesized using a nucleic acid synthesizer or the like, and can also be prepared using a cDNA display method or the like. When the cDNA is prepared by the cDNA display method, a linker for preparing an mRNA / cDNA-protein conjugate described later is used.

(2) Preparation of a linker for peptide aptamer production In the present specification, the term “linker” refers to an mRNA-linker conjugate, an mRNA-linker-protein conjugate, an mRNA / cDNA-linker-protein conjugate used in a cDNA display method. (Hereinafter, this conjugate is sometimes referred to as “IVV”), and a linker used to generate any conjugate selected from the group consisting of cDNA-linker-protein conjugate (FIG. 1).

  (L1) a solid phase binding site having a molecule that forms a bond with a solid phase; and (L2) a linker for sandwiching the solid phase binding site, and (L3) an mRNA linking site located at the 5 ′ end of the linker and recognizable by RNA ligase; and (L4) a side chain binding site located near the 3 ′ end of the linker. A main chain comprising: (L5) a primer region located on the 3 ′ end side of the linker and functioning as a primer for reverse transcription when reverse transcription is performed on the linker; and (L6) the side chain. And a side chain linked to the linking site.

The linker is mainly composed of DNA, but may include DNA analogs such as deoxyinosine, biotin-modified deoxythymine, and fluorescein-modified deoxythymine, and is designed to have flexibility and hydrophilicity as a whole. Is preferred. The term "solid phase synthesis" refers to a synthesis reaction in which the linker of the present invention is fixed directly or indirectly to beads, side walls, a bottom surface, or other solid surfaces of a reaction vessel.
In addition, in the present specification, the “predetermined mRNA” includes mRNA having a sequence encoding a gene, a sequence necessary for formation of a conjugate or promoting a translation reaction, or another sequence.

  Examples of the molecule that forms a bond with the solid phase include biotin when avidin and streptavidin and the like are bound to the solid phase, maltose when the maltose binding protein is bound, and G protein when the maltose binding protein is bound. If guanine nucleotides are bound, metals such as Ni or Co are bound when polyhistidine peptide is bound, glutathione is bound if glutathione-S-transferase is bound, sequence-specific DNA or RNA binding If a protein is bound, DNA or RNA having a sequence specific to them, an antigen or an epitope peptide if an antibody or aptamer is bound, a calmodulin-binding peptide if calmodulin is bound, ATP, estradiol when ATP binding protein is bound When a receptor protein is bound, estradiol and the like can be mentioned. Among these, it is preferable to use metals such as biotin, maltose, Ni or Co, glutathione, antigen molecules or epitope peptides, and biotin is often used from the viewpoint of ease of linker synthesis.

  The solid phase binding site is a site for binding a conjugate such as the above-described mRNA-linker-protein conjugate to a solid phase via a linker, and is composed of at least 1 to 10 bases. The solid phase binding site may contain, for example, biotin-modified deoxythymidine (dT).

  The mRNA linking site of (L3) is composed of at least 1 to 10 bases. The mRNA linking region does not need to be phosphorylated in advance, but in order to be linked to a predetermined mRNA, a kinase or the like may be used prior to or during the ligation reaction with the 3 ′ end of the mRNA. It needs to be phosphorylated.

  The side chain binding site of (L4) located near the 3 'end of the linker is a site to which a side chain described below is connected. Further, for example, when the side chain linking site of the linker is composed of Amino-Modifier C6 dT, the 5 ′ end of the side chain is 5′-Thiol-Modifier C6, and cross-linked using EMCS, The main chain and the side chain can be linked.

The primer region (L5) is located on the 3 ′ end side of the linker, and functions as a primer for reverse transcription when reverse transcription is performed on the linker. 'Adjacent to the end. Here, the primer region is a region that functions as a primer for reverse transcription when reverse transcription is performed on the linker. This region preferably comprises about 1 to 15 bases, particularly preferably 3 to 5 bases. If the number of bases exceeds 15 bases, the binding efficiency as a linker deteriorates. Therefore, the number of bases is preferably set to the above number from the viewpoints of the binding efficiency with the linker and the reaction efficiency as a primer.
Further, the side chain linked to the side chain linking site of (L6) is a spacer between the protein linking site linking a protein synthesized from mRNA complementary to the main chain and the side chain linking site. And a fluorescent group.

  Here, as the protein linking site of the side chain, puromycin, ribocytidyl puromycin (rCpPur), deoxydilpuromycin (dCpPur), deoxydyl puromycin (dCpPur) in which an amino acid forms an amide bond with the 3 ′ end of the nucleotide sequence. Puromycin analogs such as uridyl puromycin (dUpPur), as well as 3'-N-aminoacylpuromycin aminonucleosides (PANS-amino acids), 3'-N-aminoacyladenosine aminonucleosides (AANS-amino acids) and the like. Can be.

  Examples of the PANS-amino acid include PANS-Gly, PANS-Val, and PANS-Ala, and examples of the AANS-amino acid include AANS-Gly, AANS-Val, and AANS-Ala. Further, those in which a nucleoside and an amino acid are ester-linked can be used, but use of puromycin is particularly preferable because the stability of protein connection at the protein connection site is high.

  Further, Spacer 18 Phosphoramidite is preferably used as the spacer because of its flexibility and low steric hindrance. In order to prevent the occurrence of steric hindrance when the puromycin analog is incorporated into the ribosome, the side chain preferably has a structure in which about 1 to 8 phosphoramidite molecules are linked, and the synthesis efficiency of the linker and the aforementioned It is more preferable that the phosphoramidite molecule has a structure in which about four such phosphoramidite molecules are linked from the viewpoint of the balance with the formation efficiency of each linked body.

  Since the side chain has a fluorescent group between the protein linking site and the side chain linking site, the presence or absence of a linker can be easily detected in each step of the cDNA display method described below. . The fluorescent group has, for example, a carboxyl group convertible to an active ester, a hydroxyl group convertible to a phosphoramidide, or a free functional group such as an amino group, and can be linked to a linker as a labeled base. It is preferable to use a fluorescent compound that can be used. Examples of such a fluorescent compound include fluorescein isothiocyanate (FITC), phycobiliprotein, rare earth metal chelate, dansyl chloride, tetramethylrhodamine isothiocyanate, and fluorescein-dT. Among these, it is preferable to use fluorescein-dT used as a compound for molecular labeling, because the synthesis is easy.

Hereinafter, a method for producing a linker for producing a peptide aptamer will be described.
First, DNA is synthesized according to a conventional method so as to have a desired sequence, and a single-stranded oligomer to be used as a main chain is prepared. As described above, the single-stranded oligomer thus synthesized has a solid-phase binding site, two or more cleavage sites, an mRNA linking site, a side chain linking site, and a primer region. The length of the single-stranded oligomer to be the main chain is appropriately determined according to the size of the two or more cleavage sites and the position in the main chain. Next, a side chain having a desired length is synthesized and linked to a side chain linking site on the main chain. For example, puromycin is introduced into the free end of the side chain, and the above-mentioned Fluorescein-dT is introduced into a fluorescent labeling site, whereby the linker for preparing the mRNA / cDNA-protein conjugate of the present invention can be obtained.

  In designing the main chain, coding sequences of various mRNAs can be referred to. Examples thereof include mRNA encoding various receptor proteins whose sequences are known, mRNA encoding various antibodies or fragments thereof, and other mRNAs. An aminoacyl-tRNA 3′-terminal analog such as puromycin or an analog thereof is incorporated into the C-terminus of a polypeptide chain produced by translation from the mRNA coding sequence, and the polypeptide chain is linked to the mRNA-linker conjugate. To do so, a sequence that does not contain a stop codon is selected. Such mRNA can be obtained by using an in vitro transcription reaction, chemical synthesis, extraction from living organisms, cells, or microorganisms, and other various methods. High reaction efficiency of cell translation.

  Further, it is preferable from the viewpoint of protein synthesis efficiency that the compound has at least one of a 7-methylated guanosine cap structure at the 5 'end and a poly A tail structure at the 3' end. It is more preferable to have a Kozak sequence or a Shine-Dalgarno sequence because it promotes the initiation of translation. The length of the mRNA used herein depends on the length of the coding region defined by the length of the protein or polypeptide to be molecularly evolved using the present invention in principle. It is preferably from 50 to 1,000 bases in terms of reaction efficiency, and more preferably from 200 to 500 bases, since the highest reaction efficiency can be obtained.

  When generating the mRNA / cDNA-linker-protein conjugate, first, the above-described mRNA / cDNA-protein conjugate preparation linker, and an mRNA having a sequence complementary to the main chain of the linker are converted to T4 RNA at the mRNA connection site. Ligation by ligase to produce mRNA-linker conjugate. Next, a protein is synthesized from the mRNA in a cell-free translation system, and the synthesized protein is linked to the protein linking site in the mRNA-linker link to generate an mRNA-linker-protein link.

  Subsequently, the mRNA-linker-protein conjugate is bound to the solid phase via the solid-phase binding site, and the solid phase bound to the mRNA-linker-protein conjugate is washed with a first buffer. Thereafter, a reverse transcription reaction is carried out using the 3 'end of the main chain as a reaction starting point and the mRNA as a template to synthesize a cDNA chain, thereby obtaining an mRNA / cDNA-linker-protein conjugate. Next, the solid phase to which the mRNA / cDNA-linker-protein conjugate is bound is washed with a second buffer, and the cleavage site of the main chain is cleaved with the predetermined endoribonuclease.

  The ligation reaction is performed at 20 μL to 40 μL in view of reaction efficiency, and the RNA: linker is performed in a molar ratio of 3: 1 to 1: 6. In order to increase the reaction efficiency and minimize the residue, the ratio is preferably 1: (1-2) (10 pmol to 20 pmol linker for 10 pmol mRNA).

  Next, using a heat block, an aluminum block, a water bath or other heating equipment, the sample solution is heated at about 60 ° C to 100 ° C for about 2 minutes to about 60 minutes, and then at room temperature for about 2 minutes to Let stand for about 60 minutes and let the temperature drop gently. Thereafter, it is further cooled to about -5C to about 10C. Specifically, for example, a sample solution is heated on an aluminum block at 90 ° C. for 5 minutes, then transferred to an aluminum block at 70 ° C. and placed for 5 minutes, and then a ligation reaction between the mRNA and the linker is performed. . For example, about 0.1 U / μL to about 2.5 U / μL T4 polynucleotide kinase, about 0.4 U / μL to about 5 U / μL T4 RNA ligase, about 2 mM to about 50 mM magnesium chloride, about 2 mM to about 50 mM DTT In a 10 mM to 250 mM Tris-HCl buffer (pH 7.0 to 8.0) containing about 0.2 mM to about 5 mM ATP, and react at about 10 ° C. to about 40 ° C. for about 1 minute to about 60 minutes. From the viewpoints of working efficiency and reaction efficiency, the reaction is preferably performed at 20 ° C. to 30 ° C. for 5 minutes to 30 minutes, more preferably at 25 ° C. for 15 minutes.

  It is preferable to use a lysate of mammalian reticulocyte cells as the cell-free translation system, and it is more preferable to use a lysate of reticulocyte cells obtained from rabbit blood. In addition, when acetylphenylhydrazine is previously administered to the mammal to induce hemolytic anemia and the like and blood is collected after several days, the ratio of reticulocytes in the blood can be increased. For example, the lysate obtained by degrading cell-derived mRNA using micrococcal nuclease, adding calcium ether diaminetetraacetic acid (EGTA) to chelate calcium, and inactivating the nuclease (hereinafter referred to as “micrococcal lysate”). Nuclease-treated ”).

For example, a reaction solution containing about 16 mM to about 400 mM potassium acetate, about 0.1 mM to about 2.5 mM magnesium acetate, about 0.2 mM to about 50 mM creatine phosphate, and 0 mM to about 0.25 mM amino acid (all of which have a concentration of A translation reaction can be performed by adding micrococcal nuclease-treated rabbit reticulocyte lysate and the above-mentioned conjugate in 10 μL to 100 μL of the final concentration.
From the viewpoint of the reaction efficiency, the amount of the rabbit reticulocyte lysate is about 8.5 μL to about 17 μL, the amount of the conjugate is about 1.2 pmol to about 2 pmol, and the size of the reaction system is about 12.5 μL to about 25 μL, and about 20 ° C. Perform for about 10 to about 30 minutes at ~ 40 ° C. The reaction solution used in this case contains about 80 mM potassium acetate, about 0.5 mM magnesium acetate, about 10 mM creatine phosphate, about 0.025 mM methionine and leucine, respectively, about 0.05 mM methionine and an amino acid other than leucine. . Translation at about 30 ° C for about 20 minutes results in high production and work efficiency.

  After the translation reaction, the protein as the translation product and the mRNA-linker conjugate are combined with, for example, about 0.3 M to about 1.6 M of potassium chloride and about 40 mM to about 170 mM of magnesium chloride (the concentrations are all final concentrations). When the reaction is performed at about 27 ° C. to about 47 ° C. for about 30 minutes to about 1.5 hours, the protein can be efficiently bound to the conjugate.

  Examples of the solid phase on which the mRNA-linker-protein conjugate is immobilized include beads such as styrene beads, glass beads, agarose beads, sepharose beads, and magnetic beads; glass substrates, silicon (quartz) substrates, plastic substrates, and metals. Substrates such as substrates (for example, gold foil substrates); containers such as glass containers and plastic containers; and membranes made of materials such as nitrocellulose and polyvinylidene fluoride (PVDF). When the solid phase is made of a plastic material such as styrene beads or a styrene substrate, a part of the linker may be directly covalently bonded to the solid phase using a known technique, if necessary. Qiagen, LiquiChip Applications Handbook, etc.). When biotin or an analog thereof is bound to the conjugate, if the avidin is bound to the solid phase, the conjugate can be easily bound to the solid phase.

  The conjugate that did not bind to the solid phase was 1 mM to 100 mM Tris-HCl containing about 0.1 M to about 10 M sodium chloride, about 0.1 mM to about 10 mM EDTA, about 0.01% to about 1% surfactant. Using a buffer (pH 7.0 to 9.0) or a phosphate buffer (pH 7.0 to 9.0) or the like, wash and remove. Use of 20 mM Tris-HCl buffer (pH 8.0) containing 2 M sodium chloride, 2 mM EDTA, and 0.1% Triton X-100 provides good washing efficiency.

  Using the 3 'end of the main chain as a reaction initiation point and using the mRNA as a template, a reverse transcription reaction is performed under predetermined conditions to synthesize a cDNA chain. As a result, an mRNA / cDNA-linker-protein conjugate is obtained. The reverse transcription reaction system can be arbitrarily selected, but the mRNA-linker protein conjugate, dNTP Mix, DTT, reverse transcriptase, standard solution, and RNase-free water (hereinafter referred to as “RNase-free water”) ) Is added to prepare a reaction system, and reverse transcription is preferably performed in this system at 30 ° C to 50 ° C for 5 to 20 minutes.

  Subsequently, washing is performed with the same buffer solution as above to remove the free mRNA / cDNA-linker-protein conjugate. Thereafter, the cleavage site of the main chain is cleaved with the endonuclease described above. As described above, various proteins can be obtained using the linker of the present invention, and cDNAs corresponding to the proteins can also be obtained.

  In the above method, the obtained mRNA and linker DNA having puromycin are immobilized on magnetic beads modified with streptavidin using an avidin-biotin bond, and the mRNA is converted from the mRNA using a cell-free translation system. Proteins are synthesized and cDNA is synthesized from mRNA using reverse transcription reaction. Therefore, the phenotypic protein and the genotype DNA sequence information are stably associated one-to-one on the magnetic beads.

  Subsequent to the cleavage / separation step, mRNA / cDNA-linker-protein conjugate can be selected by utilizing the affinity of the obtained protein and the like. Thereafter, a mutation is introduced into the selected base sequence in the ligated product using a PCR method or the like, and an amplification reaction is performed. The amplification product is ligated to a double-stranded DNA having a desired promoter sequence by a predetermined method to obtain a first-generation mutant mRNA (hereinafter abbreviated as “mRNA G1”). Then, by using the mRNA G1, by repeating the above steps of the cDNA display method, mRNA G2, mRNA G3, etc. can be obtained, by repeating the above reaction, by introducing a desired mutation and the like Thus, peptide aptamers having various sequences can be obtained.

(3) Preparation of Chemically Cross-Linked Peptide Using a linker prepared as described above, a peptide having a desired base sequence is prepared, and a peptide in which a disulfide bond contained in the peptide is chemically cross-linked is prepared. This will be described in detail.

  The magnetic beads used in the present invention are not particularly limited as long as the magnetic beads respond to magnetism. For example, particles having a magnetic substance such as magnetite, γ-iron oxide, and manganese zinc ferrite can be used. The magnetic gel beads used in the present invention are preferably in a dispersed state even after binding to the target molecule. For this reason, the average particle size is preferably from 50 nm to 200 nm, and more preferably about 100 nm. The magnetic beads preferably have immobilized molecules for immobilizing the peptide aptamer on the surface thereof. Examples of such immobilized molecules include biotin and avidin.

  The method for synthesizing a chemically crosslinked peptide is summarized as follows. First, a template DNA is prepared, transcribed to obtain mRNA, and the obtained mRNA is linked to puromycin linker DNA. Next, after performing cell-free translation to obtain an mRNA-linker-protein conjugate, the conjugate is immobilized on magnetic beads. After immobilization, the mRNA of this conjugate is degraded, disulfide bonds in the expressed peptide molecule are reduced, and a chemical crosslink is formed between the reduced thiol groups. After biotinylation of a thiol group that has not been chemically crosslinked in the expressed peptide molecule, the thiol group is recovered from the magnetic beads and purified using a His-tag.

  Specifically, a desired peptide can be used as a template DNA as described below. For example, when a receptor for interleukin 6 (Interleukin 6 Receptor, hereinafter sometimes referred to as “IL-6R”) is selected as a target protein (peptide) used in the evaluation after peptide synthesis, IL-6R is used. It is preferable to use, as a template, a DNA encoding the S1-Cys2-6 peptide (J. Yamaguchi, et al., Nucleic Acid Res., 37, e108, (2009)) that binds to S. The S1-Cys2-6 peptide has two cysteine residues and can form a cyclic structure by forming a disulfide bond. Therefore, by selecting such a combination, the binding ability of the synthesized peptide can be accurately evaluated. The amino acid sequence and the base sequence of the S1-Cys2-6 peptide are shown as SEQ ID NOS: 1 and 2 in the sequence listing, respectively.

(Amino acid sequence of S1-Cys2-6 peptide)
MGCYPFVIAVHFSPGHSLRYPASYCNDASASTLFIGTY

(Base sequence of S1-Cys2-6 peptide)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGGCTGTTATCCTTTCGTGATCGCCGTGCACTTCGCTCTCCTGGCCACTCTCCATGCGATGATTTCTGTCGATCGATGATTTCTACGTCGATGTCATTGCTAGCGATCACGG

  The DNA sequence represented by SEQ ID NO: 2 can include a desired sequence at the 5 'end and the 3' end. For example, it has a T7 promoter sequence, a Cap sequence, an Ω sequence (translation enhancer of tobacco mosaic virus) and a Kozak sequence for translation initiation at the 5 ′ end, and has, for example, an x6 histidine tag (hereinafter, “His-tag”) at the 3 ′ end. And a tag sequence complementary to a part of the linker DNA.

  Transcription is performed using the template DNA having the above structure. For such transcription, for example, a commercially available transcription kit can be used, and it is preferable to use a Promega kit (RiboMAX Large Scale RNA Production Systems-T7) because transcription can be performed quickly and easily. When using such a kit, transcription may be performed on a scale of 10 μL to 30 μL using, for example, 0.5 μg to 5 μg of dsDNA according to the protocol attached to the kit. Using a commonly used thermostat, incubation is performed under desired conditions, a desired amount of DNase is added, and incubation is performed again to synthesize mRNA.

  For example, using an aluminum block thermostat (Anatech, Cool Stat 5200), incubate at 35 ° C. to 40 ° C. for 1 hour to 3 hours, and then add 0.5 μL to 2 μL of RQ1 DNase included in the kit to the sample. In addition, it is preferable from the viewpoint of synthesis efficiency that mRNA is further obtained by incubating at 35 ° C to 40 ° C for 5 minutes to 30 minutes. The obtained mRNA can be purified using a desired kit. As such a purification kit, for example, an After Tri-Reagent RNA Clean-Up Kit (Favogen) can be used.

  Next, an RNA ligase buffer, BSA and nuclease-free water are added to prepare a binding reaction solution. For example, a binding reaction solution is prepared by adding 5 to 15 × T4 RNA ligase buffer, 0.05% to 0.2% BSA and nuclease-free water. For example, 10 pmol to 30 pmol of the mRNA transcribed as described above is added, a binding reaction liquid is added at 12 to 24 μL, the mixture is incubated under desired conditions, nucleotide kinase and RNA ligase are added in desired amounts, and again under desired conditions. By incubating, an mRNA-linker conjugate can be obtained.

  As the pull-down linker used here, one having an mRNA binding site and a solid phase binding site is preferably used because the peptide synthesized later can be easily purified. For example, it is preferable to use a linker having the sequence of SEQ ID NO: 3 in the sequence listing as the pull-down linker.

  Specifically, for example, the above reaction solution is incubated at 80 ° C. to 95 ° C. for 0.5 minutes to 2 minutes, and then incubated at 60 ° C. to 80 ° C. for 0.5 minutes to 2 minutes, and then 0.02 ° C./s to 0.06 ° C. / Cool down to 20-30 ° C at s speed. Thereafter, for example, T4 polynucleotide kinase (manufactured by TaKaRa) in an amount of 0.25 μL to 0.75 μL, and T4 RNA ligase (manufactured by TaKaRa) in an amount of 0.5 μL to 2 μL are added to the reaction solution, respectively, and the mixture is added at 20 ° C. to 30 ° C. By incubating for 0.5 to 2 hours, an mRNA-linker conjugate can be obtained.

Thereafter, the mRNA-linker conjugate obtained as described above is placed in a tube having a desired capacity, and cell-free translation is performed under desired conditions. By adding MgCl ₂ and KCl in desired amounts and incubating under desired conditions, an mRNA-linker-peptide conjugate (hereinafter sometimes referred to as “mRNA-linker-protein conjugate”) can be obtained. . As the cell-free translation system, commercially available products such as Retic Lysate IVT Kit (manufactured by Ambion) can also be used.

For example, the mRNA-linker conjugate is translated using a cell translation system of 40 μL to 60 μL per 4 pmol to 8 pmol. For example, in a plurality of 1.5 mL to 2 mL tubes, the above-described mRNA-linker conjugate is added, and a desired amount of the cell-free translation system, for example, 20 μL to 30 μL is dispensed at 25 ° C. to 35 ° C. for 15 minutes. Incubate for ~ 30 minutes. Thereafter, MgCl ₂ and KCl are added to a final concentration of 60 mM to 100 mM and 600 mM to 1,000 mM, respectively, and incubated at 35 ° C. to 40 ° C. for 0.5 to 2 hours to obtain an mRNA-linker-protein conjugate. It is preferable from the viewpoint of efficiency.

  Next, the mRNA-linker-peptide conjugate is immobilized on magnetic beads. For example, as such magnetic beads, commercially available beads such as Dynabeads MyOne Streptavidin C1 can be used. First, the conjugate is put together in an appropriate amount, a binding buffer is added, and the mixture is incubated under desired conditions to remove ribosomes bound to the conjugate. For example, the above-mentioned conjugates are put together for each two tubes to make a solution volume of about 60 μL to 100 μL, and 10 μL to 25 μL of 0.25 M to 1 M EDTA (pH 8.0) and 90 μL to 100 μL of 1 to 4 × binding buffer (10 mM to 25 mM) Tris-hydrochloric acid (pH 7.5), containing 1.5 M to 3 M NaCl, 1 to 3 mM EDTA, and 0.1% to 0.5% Tween-20) and incubating at 2 ° C to 6 ° C for 5 minutes to 15 minutes. Then, the ribosome bound to the mRNA-linker-protein conjugate is removed to obtain a translation product. The total volume of the solution is between about 180 μL and 200 μL.

  Subsequently, the magnetic beads washed with the 1x binding buffer are put into a tube of a predetermined size, the above-mentioned translation product is added, the mixture is incubated under desired conditions, and a protein for ligation is added and incubated under the predetermined conditions. As such magnetic beads, for example, Dynabeads MyOne Streptavidin C1 (invtrogen) coated with streptavidin can be used.Preferably, 20 μL to 40 μL of the above beads are put in a 1.5 mL to 2 mL tube, and placed here. Add 180 μL-200 μL of the translation product and stir at 20-30 ° C. for 15-45 minutes.

  At this time, for example, a cooling thermoblock rotator (SNP-24B, manufactured by Nissin Chemical Co., Ltd.) can be used. Thereafter, biotin is added to a final concentration of 5 μM to 15 μM, and the mixture is further stirred at 20 ° C. to 30 ° C. for 10 minutes to 20 minutes. When the magnetic beads are coated with biotin instead of streptavidin, streptavidin may be added last.

  Next, the mRNA of the mRNA-protein conjugate (hereinafter sometimes referred to as “mRNA-peptide conjugate”) is degraded. The magnetic beads having the mRNA-linker-protein conjugate immobilized thereon are washed a desired number of times using a desired amount of a binding buffer. Next, washing is performed with a desired amount of NE buffer, the above NE buffer containing a desired amount of RNase H is added, and the mixture is stirred under desired conditions to decompose mRNA to obtain a linker-protein conjugate.

  The beads on which the conjugate has been immobilized are washed 1-3 times with 75 μL to 150 μL of 2 × binding buffer, and then, for example, once with 25 μL to 75 μL of 1 × NE buffer 2 (BioLabs). Thereafter, for example, 25 to 75 μL of 1 × NE buffer 2 containing 10 to 30 U of RNase H (manufactured by TaKaRa) is added, and the mixture is stirred at 35 ° C. to 40 ° C. for 15 minutes to 45 minutes using the above-mentioned cooled thermoblock rotator or the like. To degrade the mRNA to obtain a magnetic bead-immobilized linker-protein conjugate.

  Next, disulfide bonds in the protein (expressed peptide molecule) immobilized on the magnetic beads are reduced. The magnetic bead-immobilized linker-protein conjugate is washed with a desired amount of a conjugation buffer, and subsequently, a conjugation buffer containing dithiothreitol (hereinafter, sometimes referred to as “DTT”) is added, and the desired conditions are added. To reduce disulfide bonds in this protein molecule.

  For example, the conjugation buffer can have a composition comprising 50 mM to 150 mM sodium phosphate, 100 mM to 200 mM NaCl, 5 mM to 15 mM EDTA, and 0.01% to 1% Tween-20. Thereafter, 150 μL to 250 μL of a 1 × conjugation buffer containing a predetermined concentration, for example, 10 mM to 30 mM dithiothreitol (hereinafter, sometimes referred to as “DTT”) is added, and the mixture is added at 23 ° C. to 28 ° C. for 15 minutes. The mixture is stirred for 25 minutes to reduce disulfide bonds in the expressed peptide molecule.

  Thereafter, a chemical crosslink is formed between the thiol groups in the expressed peptide molecule. The magnetic beads thus reduced are washed with the conjugation buffer, and then the conjugation buffer containing the desired cross-linking compound is added and stirred under predetermined conditions to form chemical cross-links. FIG. 3 shows a schematic diagram of the procedure. Specifically, after the reduction, the magnetic beads on which the above-mentioned expressed peptide is immobilized are washed once or twice with 50 μL to 200 μL of 1 × conjugation buffer, and 100 μM to 300 μM of bis (maleimide) ethane (hereinafter, “BMOE”). ) Is added, and the mixture is stirred at 20 ° C to 30 ° C for 0.5 hours to 2 hours to form a chemical crosslink, thereby obtaining a crosslinked peptide.

Subsequently, a thiol group that has not formed a chemical crosslink in the above expressed peptide molecule is biotinylated. Again, the above-described reduction operation is performed to reduce disulfide bonds that have not formed a chemical crosslink. Thereafter, the magnetic beads are washed with the conjugation buffer, and then reacted with the reduced disulfide under a desired condition using a desired concentration of a compound for biotinylation for biotinylation. As such a compound for biotinylation, maleimide-PEG ₁₁ -biotin (manufactured by Thermo scientific) or the like can be used. More specifically, after the above reduction operation, and washing the magnetic beads 1x conjugation buffer 50Myueru～100myuL, for example, maleimide-PEG ₁₁ of 100Myueru～300myuL - adding 1x conjugation buffer containing biotin Stir at 20-30 ° C for 2-3 hours.

  After completion of the stirring, the chemically cross-linked peptide is recovered from the magnetic beads in a state of being bonded to the linker (as a linker-protein conjugate). First, the magnetic beads are washed with a desired washing buffer, and then a releasing agent is added and incubated to release the peptide from the magnetic beads. Examples of such a washing buffer include 1 × His-tag washing buffer (containing 10 to 30 mM sodium phosphate (pH 7.4), 0.25 M to 0.75 M NaCl, 2.5 mM to 10 mM imidazole, and 0.025 to 0.1% Tween-20). Can be used. As the releasing agent, a 1 × His-tag washing buffer containing a desired concentration of RNase, for example, 500 U to 1,500 U of RNase T1 can be used.

  For example, the magnetic beads are washed 1 to 3 times with 50 μL to 200 μL of the 1 × His-tag buffer, and then the RNase T1-containing 1 × His-tag washing buffer is added, and the mixture is added at 35 ° C. to 40 ° C. for 5 to 15 minutes. Incubate. The supernatant is collected and the chemically crosslinked peptide is recovered as a linker-protein conjugate.

  The resulting chemically crosslinked peptide is purified using His-tag protein purification beads. As such beads for His-tagged protein purification, His Mag Sepharose Ni (manufactured by GE healthcare) or the like can be used. The beads are washed in advance with the His-tag washing buffer. Next, the recovered chemically crosslinked peptide was added to the washed beads for purifying the His-tagged protein, incubated under desired conditions, and the beads were washed with the His-tagged washing buffer. (Hereinafter, sometimes referred to as “His-tag elution buffer”) and stirred under desired conditions to obtain a purified chemically crosslinked peptide. As such a His-tag elution buffer, a buffer containing 10 mM to 30 mM sodium phosphate (pH 7.4), 0.25 to 0.75 M NaCl, 100 mM to 500 mM imidazole, and 0.025 to 0.1% Tween20 can be used.

  For example, when His Mag Sepharose Ni (manufactured by GE healthcare) washed with 1 × His-tag washing buffer, about 1/5 of the collected linker-protein conjugate, for example, when the collected amount is 50 μL to 200 μL , Add 10 μL to 40 μL, and stir at 20 ° C. to 30 ° C. for 0.5 to 2 hours using a cooling thermoblock rotator or the like. Thereafter, for example, washing is performed 2 to 4 times using 50 μL to 200 μL of 1 × His-tag washing buffer, 5 μL to 20 μL of His-tag elution buffer is added, and a micro tube mixer (manufactured by Tommy Seiko, MT-360) is added. Use and stir at room temperature for 10-20 minutes. By the above operation, the produced chemically crosslinked peptide can be obtained.

  Further, the chemically crosslinked peptide obtained as described above is immobilized on magnetic beads, and the beads are reacted with a sample under desired conditions to screen for proteins that interact with these peptides. Can be. At this time, if the luminophore is bound to the magnetic beads so that light is emitted when the chemically crosslinked peptide and the target protein are bound, the detection is further facilitated.

  Furthermore, by introducing a mutation into the above-described template according to a conventional method, a very large number of peptides that can bind to IL-6R can be obtained. Then, a peptide library targeting IL-6R can be obtained by chemically cross-linking such a peptide by the above-described method.

(Example 1) Preparation of a linker for cDNA display (1) Reagents Modified oligonucleotides, Puro-FS and biotin loop, were obtained from Gene World (Tokyo). A Puro-FS having a structure of 5 ′-(S) -TC (F)-(Spec18)-(Spec18)-(Spec18)-(Spec18) -CC- (Puro) -3 ′ was obtained. In this Puro-FS chain, (S) represents 5'-Thiol-Modifier C6, (F) represents fluorescein-dT, (Puro) represents puromycin CPG, and (Spacer18) represents spacer phosphoramidite 18. . As the biotin loop, an oligonucleotide having the following sequence (SEQ ID NO: 3 in the sequence listing) was used.

(Base sequence of oligonucleotide used as biotin loop)
5'-CCCGGTGCAGCTGTTTCATC (TB) CGGAAACAGCTGCACCCCCCGCCGCCCCCCG (T) CCT-3 '

  In the biotin loop chain, (T) represents Amino-Modifier C6 dT, and (T-B) represents Biotin-dT (underlined represents a recognition site for PvuII restriction enzyme). EMCS [N- (6-maleimidocaproyloxy) succinimide] was purchased from Dojin Chemical Research Laboratories (Kumamoto, Japan). TCEP [tris (2-carboxyethyl) phosphine was purchased from Pierce.

(2) Synthesis of puromycin linker DNA The 5 ′ thiol group of the above Puro-FS (10 nmol) was reduced with 1 mM TCEP in 100 μL of 50 mM phosphate buffer (pH 7.0) for 6 hours at room temperature. Then, desalting was performed as necessary using a NAP-5 column (manufactured by GE Healthcare). A total of 10 nmol of biotin loop and 2 μmol of EMCS were added to 100 μL of 0.2 M sodium phosphate buffer (pH 7.0). Subsequently, the mixture was incubated at 37 ° C for 30 minutes and ethanol precipitated at 4 ° C to remove excess EMCS.

This precipitate was washed twice with 500 μL of 70% ethanol which had been cooled beforehand, and dissolved in 10 μL of 0.2 M sodium phosphate buffer (pH 7.0) which had been cooled previously. The reduced Puro-FS was immediately added and stirred at 4 ° C. overnight. The reaction was stopped by adding 4 mM TCEP and incubating at 37 ° C. for 15 minutes. Thereafter, ethanol precipitation was performed to remove excess Puro-FS at room temperature. In order to remove the biotin loop and the uncrosslinked biotin loop-EMCS complex, the precipitate is dissolved in 0.1 M TEAA (manufactured by Glen Research) or phosphate buffer and purified using a _C18 HPLC column under the following conditions. did.

Column: AR-300, 4.6x250mm (Nacalai Tesque, Japan)
Solvent: solvent A: 0.1 M TEAA; solvent B: acetonitrile / water (80:20, v / v)
Gradient: B / A (15-35%, 33 minutes)
Flow rate: 0.5mL / min Detection wavelength: Absorbance 254nm and 490nm

  Fractions from the fraction from the last peak at 254 nm absorbance (corresponding to a single peak at 490 nm absorbance) were collected. After drying, it was resuspended in diethylpyrocarbonate (DEPC) treated water and stored. As described above, puromycin-linker DNA was obtained.

Example 2 Preparation of Linker-Protein Conjugate (Step 1) Preparation of Template DNA The template DNA used is an S1-Cys2-6 peptide that binds to the interleukin-6 receptor (IL-6R). (SEQ ID NO: 1 and FIG. 2a). This peptide has two cysteine residues and forms a disulfide bond, and thus has a characteristic of taking a cyclic structure.

(S1-Cys2-6 amino acid sequence)
MGCYPFVIAVHFSPGHSLRYPASYCNDASASTLFIGTY

  The DNA used in this example (the following sequence, shown by SEQ ID NO: 2) has a T7 promoter sequence, Cap, Ω sequence, and Kozak sequence at the 5 ′ end, and His-tag and linker at the 3 ′ end. It has a tag sequence complementary to a part of DNA. These arrangements in the template DNA are summarized in Table 1 below.

(Base sequence of template DNA)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGGCTGTTATCCTTTCGTGATCGCCGTGCACTTCGCTCTCCTGGCCACTCTCCATGCGATGATTTCTGTCGATCGATGATTTCTACGTCGATGTCATTGCTAGCGATCACGG

(Step 2) Transcription of Template DNA Transcription was performed on a 20 μL scale using 1 μg of dsDNA according to the protocol attached to the Promega kit (RiboMAX Large Scale RNA Production Systems-T7). Incubation was performed at 37 ° C for 2 hours using an aluminum block thermostat (Anatech, Cool Stat 5200). Thereafter, DNase (RQ1 DNase) included in the kit was added to 1 μL of the sample, and further incubated at 37 ° C. for 15 minutes. The synthesized mRNA was purified using the After Tri-Reagent RNA Clean-Up Kit (Favogen) (FIG. 2a).

(Step 3) Binding of mRNA to puromycin linker DNA Per 20 pmol of mRNA obtained by transcription, 22.5 pmol of pull-down linker DNA (SEQ ID NO: 3), 2 μL of 10 × T4 RNA ligase buffer (TaKaRa), and 1.2 μL of 0.1% BSA was added thereto, and a reaction solution for binding was prepared so that the total volume was 18.5 μL with nuclease-free water. The structure of the pull-down linker represented by SEQ ID NO: 3 in the sequence listing is schematically shown in FIG. The above reaction solution was incubated at 90 ° C. for 1 minute, then at 70 ° C. for 1 minute, and then cooled to 25 ° C. at a speed of 0.04 ° C./s. 0.5 μL of T4 polynucleotide kinase (TaKaRa) and 1 μL of T4 RNA ligase (TaKaRa) were added to the above reaction solution, and the mixture was incubated at 25 ° C. for 1 hour to obtain an mRNA-linker conjugate (FIG. 2 a). ).

(Step 4) Translation Using a Cell-Free Translation System A translation of 6 μmol of the mRNA-linker conjugate obtained as described above was translated on a 50 μL scale using a cell-free translation system (Retic Lysate IVT Kit, manufactured by Ambion). . The above mRNA-linker conjugate was dispensed in 25 μL aliquots into 1.75 mL tubes, and then translated at 30 ° C. for 20 minutes. Thereafter, MgCl ₂ and KCl were added to a final concentration of 80 mM and 800 mM, respectively, and incubated at 37 ° C. for 1 hour to obtain an mRNA-linker-protein conjugate (FIG. 2 a).

(Step 5) Immobilization on Magnetic Beads Translated products are combined into two tubes (liquid volume is 78 μL), and 18 μL of 0.5 M EDTA (pH 8.0), 96 μL of 2 × binding buffer (20 mM Tris-HCl) (pH 7.5), containing 2M NaCl, 2mM EDTA, 0.2% Tween-20) and incubating at 4 ° C for 10 minutes to remove ribosomes bound to the mRNA-linker-protein conjugate (liquid Volume is 192 μL).

  Then, Dynabeads MyOne Streptavidin C1 (invtrogen) was pre-washed with 1x binding buffer. 30 μL of the washed cells are previously placed in a 1.75 mL tube, and 192 μL of the translation product is added thereto. Next, the mixture was stirred at 25 ° C. for 30 minutes using a cooling thermoblock rotator (SNP-24B, manufactured by Nisshin Rika Co., Ltd.). Thereafter, biotin was added to a final concentration of 10 μM, and the mixture was stirred at 25 ° C. for 15 minutes (FIG. 2A).

(Step 6) mRNA degradation of mRNA-protein conjugate
The Dynabeads MyOne Streptavidin C1 on which the mRNA-linker-protein conjugate was immobilized was washed twice with 100 μL of 1 × binding buffer. The plate was washed once with 50 μL of 1 × NE buffer 2 (manufactured by BioLabs). Thereafter, 50 μL of 1 × NE buffer 2 containing 20 U of RNase H (TaKaRa) was added, and the mixture was stirred at 37 ° C. for 30 minutes using a cooling thermoblock rotator to obtain a linker-protein conjugate (FIG. 2 a).

(Step 7) Reduction of disulfide bond in expressed peptide molecule Dynabeads MyOne Streptavidin C1 having immobilized linker-protein conjugate was added to 100 μL of 1 × conjugation buffer (100 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, 0.05% Tween- 20). Thereafter, 200 μL of 1 × conjugation buffer containing 20 mM dithiothreitol (hereinafter, sometimes referred to as “DTT”) was added, and the mixture was stirred at 25 ° C. for 20 minutes (FIG. 2B).

(Step 8) Chemical cross-linking between thiol groups in the expressed peptide molecule The reduced Dynabeads MyOne Streptavidin C1 was washed once with 100 μL of 1 × conjugation buffer, and then 200 μM bis (maleimide) ethane (Thermo scientific) (Hereinafter, may be referred to as “BMOE”) in an amount of 200 μL and stirred at 25 ° C. for 1 hour to obtain a crosslinked product (FIGS. 2B and 3A). 2a and 2b show schematic diagrams of the above steps 2 to 8.

(Step 9) Biotinylation of an unchemically crosslinked thiol group in the expressed peptide molecule The reduction operation shown in the above step 7 was performed again to reduce disulfide bonds that were not chemically crosslinked. Thereafter, Dynabeads MyOne Streptavidin C1 was washed once with 100 μL of 1 × conjugation buffer. Next, 200 μL of 1 × conjugation buffer containing 625 μM maleimide-PEG ₁₁ -biotin (manufactured by Thermo scientific) was added, and the mixture was stirred at 25 ° C. for 2 hours and 30 minutes to obtain a reduced / biotinylated product (FIGS. 3 and 3b). ).

(Step 10) Recovery from magnetic beads
Dynabeads MyOne Streptavidin C1 was washed twice with 100 μL of 1 × His-tag washing buffer (containing 20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 5 mM imidazole, 0.05% Tween-20). Thereafter, 100 μL of 1 × His-tag washing buffer containing 1,000 U of RNase T1 (Ambion) was added, and the mixture was incubated at 37 ° C. for 10 minutes. The supernatant was collected to obtain a linker-protein conjugate (enzyme-treated product) (FIGS. 3 and 3c).

(Step 11) Purification by His-tag 100 μL of the collected linker-protein conjugate was added to His Mag Sepharose Ni (manufactured by GE healthcare) washed with 1 × His-tag washing buffer in an amount of 20 μL, and the cooled thermoblock rotator was used. Stirred at 25 ° C. for 1 hour. After washing three times with 100 μL of 1 × His-tag washing buffer, 12 μL of His-tag elution buffer (containing 20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 250 mM imidazole, 0.05% Tween 20) was added, and a micro tube mixer ( The mixture was stirred at room temperature for 15 minutes using MT-360 manufactured by Tommy Seiko to obtain a His-tag product (FIGS. 3 and 3d). FIG. 3 is a schematic diagram showing steps 8 to 11 of the second embodiment.

Example 3 Calculation of Crosslinking Efficiency of Linker-Protein Conjugate (1) Calculation of Crosslinking Efficiency The crosslinking efficiency of the linker-protein conjugate was determined using a gel shift assay method using a neutravidin protein. First, neutravidin protein was added to the purified linker-protein conjugate obtained as in steps 1 to 11 of Example 1 so that the final concentration became 60 μM, and the mixture was incubated at 25 ° C. for 30 minutes. Avidin product was obtained (FIG. 3d). This was electrophoresed by 15% SDS-PAGE (20 mA, 120 minutes), and the crosslinking efficiency was calculated from the intensity ratio of the gel-shifted band (FIGS. 4A and 4B). Crosslinking efficiency was about 90%.

(Example 4) Evaluation of function of protein obtained by pull-down method (1) Fluorescence of target protein IL-6R
1.75 mL of 1 × PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO4, 1.8 mM KH ₂ PO ₄ ) containing 1 μM IL-6R (ACRO Biosystems) and 20 μM NHS-fluorescein (manufactured by Thermo scientific) on a 50 μL scale. Tubes were incubated at 25 ° C. for 60 minutes. Thereafter, 2-aminoethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 100 μM, and the mixture was incubated at 25 ° C. for 30 minutes to crush unreacted active groups of NHS-fluorescein.

(2) Pull-down Method Using Target Molecule (IL-6R) Dynabeads MyOne Streptavidin C1, on which a linker-protein conjugate was immobilized, prepared as in Steps 1 to 8 above, was washed three times with 100 μL of 1 × PBS. did. Thereafter, 100 μL of 1 × PBS containing 100 nM of fluorescent IL-6R was added, and the mixture was stirred at 25 ° C. for 60 minutes. Next, the immobilized beads were washed three times with 1 × PBS-T (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO4, 1.8 mM KH ₂ PO ₄ , and 0.01% Tween 20), and contained 1,000 U of RNase T1. 15 μL of 1 × PBS-T was added and incubated at 37 ° C. for 10 minutes. The collected sample was run on a 15% SDS PAGE (20 mA, 120 minutes) to compare the amount of IL-6R pulled down. FIG. 5 shows a schematic diagram of the above process for reference.

  In this example, seven types of samples were prepared for comparison. IL-6R fluoresced in Example 1 was used as a control sample for the IL-6R band position. As a negative control, a sample was prepared in which steps 4 to 8 of Example 1 above were performed on the pull-down linker DNA. As a positive control, a sample in which a disulfide bond (S-S cross-linking) was combined with the Cys2-6 peptide was prepared. Furthermore, a chemically cross-linked Cys2-6 peptide and a non-cross-linked Cys2-6 peptide having a thiol group crushed were prepared (FIG. 5).

The non-crosslinked Cys2-6 peptide was subjected to the reduction operation performed in step 7 of Example 1 above, and washed twice with 100 μL of 1 × conjugation buffer. Thereafter, 200 μL of 1 × conjugation buffer containing 5 mM iodoacetamide was added, and the mixture was stirred at 25 ° C. for 20 minutes to prepare. In addition, as a model of a peptide having no interaction with IL-6R, a sample using the B domain of A protein (hereinafter sometimes referred to as “BDA”) (SEQ ID NO: 4) and FLAG (SEQ ID NO: 5) Was also adjusted. These sample preparations were performed according to the steps described above (FIGS. 5a to 5c).
The base sequence of the B domain of A protein (SEQ ID NO: 4) and FLAG (SEQ ID NO: 5) are shown below. Tables 2 and 3 below summarize the respective regions and functions in each DNA.

(Base sequence of B domain of A protein)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGATAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAACGAAGAACAACGCAATGGTTTCATCCAAAGCCTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCTCAAGCACCAAAAGCTGACAACAAATTCAACGGGGGAGGCAGCCATCATCATCATCATCACGGCGGAAGCAGGACGGGGGGCGGCGGGGAAA-3 '

(Base sequence of FLAG)
5'-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGATTATAAGGACGATGACGATAAGGGAGGTGGAGGTAAGAACATGTGCAGGAGCATGAAGCCCTGGAGCATGAGAGTCCCTGGAGGAGAGTGGCTGGAGCATAAGA

(Example 5) Evaluation of interaction by pull-down method Using the sample adjusted as described above, a pull-down method was performed under the same conditions as in Example 3, and the affinity for IL-6R was evaluated. As a result, it was confirmed from the electrophoretogram that the chemically cross-linked Cys2-6 peptide was able to interact with the same degree as the SS cross-linked Cys2-6 peptide as a positive control (FIG. 6). In addition, no interaction was shown for Cys2-6 without the disulfide bridge, confirming that the shape of the peptide was greatly related to the function of the peptide.

  As described above, it was shown that the peptide having the chemical crosslink of the present invention has the same binding ability to the target protein as the peptide having no chemical crosslink. In the case of chemical crosslinking, there is an advantage that the length of the crosslinking can be adjusted depending on the molecule used for the crosslinking.

  INDUSTRIAL APPLICABILITY The present invention is useful in the field of medicine, particularly in the field of diagnostic agents.

SEQ ID NO: 1: Amino acid sequence of S1-Cys2-6 peptide SEQ ID NO: 2: Nucleotide sequence of S1-Cys2-6 peptide SEQ ID NO: 3: Biotin loop SEQ ID NO: 4: Nucleotide sequence of B domain of A protein SEQ ID NO: 5: FLAG Base sequence

Claims (6)

A chemically crosslinked peptide having a crosslinked structure that is stable even under reducing conditions, prepared by a method for preparing a chemically crosslinked peptide comprising the following steps (a) to (e):
(A) a DNA sequence comprising a DNA sequence of a target protein, a DNA sequence containing a desired nucleotide sequence at the 5 ′ end of the DNA sequence of the target protein, and a DNA sequence containing a desired nucleotide sequence at the 3 ′ end of the DNA sequence of the target protein An mRNA preparation step of preparing an mRNA by transcription from any nucleotide sequence selected from the group;
(B) a linker preparing step of ligating the mRNA obtained in the step (a) to an mRNA binding site of a linker having a peptide binding site, a solid phase binding site and an mRNA binding site;
(C) a peptide synthesis step of translating the linker obtained in the step (b) by a cell-free translation system and synthesizing a peptide corresponding to the mRNA in a state of being bound to a peptide binding site of the linker;
(D) an immobilization step of immobilizing the linker obtained in the step (c) on a solid phase;
(E) a cross-linking compound that cuts the SS bond in the peptide bound to the linker immobilized on the solid phase in the step (d) by reduction into an -SH group, and then forms a cross-link And a chemical cross-linking step of binding the reduced —SH group to form an intramolecular cross-link of the peptide;
The cross-linking compound is a divalent cross-linking agent selected from the group consisting of bismaleimide ethane, 1,6-bismaleimide hexane, 1,4-bismaleimide butane, and 1,4-bismaleimidyl-2,3-dihydroxybutane. Is;
The above-mentioned chemically crosslinked peptide has a crosslinking efficiency of 90% or more with a linker by a gel shift assay.   The chemically crosslinked peptide according to claim 1, wherein the peptide binding site is composed of any one selected from the group consisting of puromycin, AANS-amino acid, and PANS-amino acid.   3. The chemically crosslinked peptide according to claim 1, wherein the solid phase binding site is composed of one selected from the group consisting of streptavidin and biotin. 4.   The chemical cross-linking peptide according to any one of claims 1 to 3, wherein the chemically cross-linking peptide has at least one cross-linking site, and has an outer diameter of 60 to 100 nm after cross-linking. Cross-linked peptide.   A method for purifying an interacting protein, comprising using the chemically crosslinked peptide having a crosslinked structure that is stable even under reducing conditions according to any one of claims 1 to 4 to purify proteins that interact with these peptides.   A method for screening interacting proteins, comprising using the chemically crosslinked peptide having a stable crosslinked structure even under reducing conditions according to any one of claims 1 to 4 to screen for proteins that interact with these peptides.

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