Ribosome display complex and production method therefor

[0104] (1) Preparation process of ribosome display complex

[0105] In this step, a cell-free peptide synthesis system using ribosomes is used to translate mRNA molecules to obtain a ribosome complex including unmodified polypeptide chains, mRNA molecules, and ribosomes.

[0106] The cell-free peptide synthesis system using ribosomes is a system that synthesizes polypeptides from mRNA in vitro using compounds required for polypeptide synthesis based on RNA information performed in cells. Specifically, mRNA molecules are added to the reaction system including proteins, ribosomes, tRNAs, amino acids, NTP, buffers, etc. required for the translation and energy regeneration of mRNA such as initiation factor, elongation factor, aminoacyl tRNA synthetase, etc. , Synthesize the polypeptide corresponding to the added mRNA. There are commercially available kits for synthesizing cell-free peptides, so it is sufficient to use commercially available kits in addition to mRNA molecules.

[0107] The ribosome display complex prepared in this step includes mRNA, a polypeptide chain as its translation product, and ribosomes. Hereinafter, ribosome display is sometimes abbreviated as "RD".

[0108] The polypeptide chain contained in the RD complex of the present invention contains selected from the group consisting of cysteine ​​residues, lysine residues, histidine residues, and tryptophan residues used for modification in the following steps One or more reactive amino acid residues in. As the number of reactive amino acid residues, there are cases where the stability of the polypeptide chain is further improved by cyclizing the polypeptide chain, and therefore, it is preferably two or more. On the other hand, when there are many reactive sites, the number and position of the modification reagents that bind to the RD complex may be unstable and it is difficult to compare with the characteristics of the polypeptide chain derived from the amino acid sequence. The number is preferably 10 or less.

[0109] As the above-mentioned reactive amino acid residue, a cysteine ​​residue and/or a lysine residue are preferable. In addition, when, for example, a cysteine ​​residue is involved in stabilizing the higher-order structure of a polypeptide through a disulfide bond, it is preferable to separately introduce the above-mentioned reactive amino acid residue.

[0110] The position of the reactive amino acid residue may be appropriately selected. For example, it is preferable that it is a part protruding to the outside from the exit tunnel of the ribosome, specifically the second position from the N-terminus to the C-terminus The position from the 30th position (including the second position from the N-terminus and the 30th position from the C-terminus). In the present invention, since the polypeptide chain is modified in the state of the RD complex, if the reactive amino acid residue is located at the above-mentioned position, the modification reaction is hardly hindered by the ribosomal space. As the position from the aforementioned C terminal, the 50th position is preferable, and the 100th position is more preferable. In addition, when counting the positions of the reactive amino acid residues from the N-terminal side, the positions can be appropriately set according to the chain length of the polypeptide, for example, the positions from the N-terminal to the 1000th position, and preferably the positions from the N-terminal. The position of 2 to 100 is more preferably the position of 2 to 50 from the N terminal.

[0111] In addition, in order to function as a polypeptide library, the polypeptide chain preferably contains a random sequence at a specific position. From the above random sequence, a useful amino acid sequence corresponding to a predetermined purpose can be determined. The position of the random sequence can also be appropriately selected. For example, it is preferable to set the position from the second position from the N-terminus to the 30th position from the C-terminus in the same way as the position of the reactive amino acid residue (including the position from the N-terminus to the 30th position). Position 2 and position 30 from the end of C). That is, the reactive amino acid residue is preferably included in a random sequence. Therefore, the preferred position of the random sequence can be set in the same range as the preferred position of the reactive amino acid residue. The number of amino acids in the random sequence can also be appropriately selected, for example, it can be set to 1 or more and 30 or less. The upper limit of the number of random sequences is not particularly limited, but it is preferably 10 or less. In addition, there may be one or more such random sequences in the polypeptide chain. The longer a random sequence and the greater the number of random sequences, the better the diversity of the peptide library.

[0112] In addition, the polypeptide chain optionally has an amino acid sequence corresponding to the purpose. For example, a sequence used for purifying a polypeptide chain such as FLAG (registered trademark) sequence and poly-His sequence, a sequence selectively cleaved by a protease, etc., a spacer sequence, etc. can be cited.

[0113] The mRNA contains at least the base sequence encoding the above-mentioned polypeptide chain. In addition, it optionally has sequences required for translation and the like. Since this mRNA encodes a polypeptide in the same RD complex, when a specific RD complex is selected from a library, the amino acid sequence of a useful polypeptide can be indirectly determined by analyzing the base sequence of the mRNA.

[0114] The number of amino acid residues in the polypeptide chain is not particularly limited. For example, it can be 100 or more and 5000 or less. The number of amino acid residues is more preferably 150 or more, still more preferably 200 or more, more preferably 800 or less or 600 or less, and still more preferably 500 or less.

[0115] Ribosomes can be purified from living organisms. For example, ribosomes derived from Escherichia coli may be used.

[0116] In intracellular polypeptide synthesis, once the polypeptide is synthesized, the dissociation factor binds to the stop codon of the mRNA, the polypeptide chain is freed, and the mRNA is also dissociated from the ribosome. However, since the purpose of this step is to prepare an RD complex containing a polypeptide chain, mRNA, and ribosomes, treatments are required to prevent the polypeptide chain from being released. As the above treatment, a known method can be used. Examples include: removing the stop codon from the mRNA, or arranging a translation extension termination sequence called an arrest sequence at the 3'end of the mRNA, or not adding a dissociation factor to the cell-free peptide synthesis system, Ribosome regeneration factor.

[0117] After the RD complex is synthesized, a conventional method may be used as a purification method of the RD complex. For example, when FLAG (registered trademark) sequence or polyHis sequence is present in the polypeptide chain, a known purification method corresponding to these sequences can be used.

[0118] (2) Modification process of polypeptide chain

[0119] After the ribosome complex containing the unmodified polypeptide chain, mRNA molecule, and ribosome is produced as described above, in this step, the side chain reactive functional group contained in the unmodified polypeptide chain is reacted with a modification reagent to control the unmodified polypeptide chain. Modify the polypeptide chain for modification. If a ribosome complex containing an unmodified polypeptide chain is produced and then reacted with a modification reagent, it is extremely simple to pass through while maintaining the state of the RD complex (that is, without impairing the binding function of the polypeptide and mRNA in the ribosome). The process of chemical modification of the polypeptide chain.

[0120] It should be noted that because ribosomes are giant molecules that are complexes of rRNA and protein, they contain a large number of reactive amino acid residues compared to the presentation polypeptide contained in the RD complex. However, even when the RD complex is made to act with a modification reagent, the presenting polypeptide chain contained in the RD complex can be modified, and the RD complex can also be maintained.

[0121] As a modification reagent for modifying the unmodified polypeptide chain, for example, a compound represented by the following formula (1) can be used.

[0122]

[0123] In the above formula, A represents a group that can react with the side chain of a cysteine ​​residue, a lysine residue, a histidine residue, or a tryptophan residue to form a bond. That is, it can interact with the sulfhydryl group of cysteine ​​residue and the side chain amino group (-NH 2 ) Or the side chain amino group (>NH) of histidine residue and tryptophan residue to form a bond.

[0124] Specifically, examples of A include halogenated alkyl groups, activated carbonyl groups, unsaturated hydrocarbon groups, epoxy groups, sulfonyl-containing groups, isocyanate groups, thioisocyanate groups, carbene generating groups, and carbene-containing groups , Disulfide bond-containing groups or mercapto groups.

[0125] Examples of the halogenated group in the halogenated alkyl group include a chloro group, a bromo group, or an iodo group. In addition, the alkylene group in the haloalkyl group may be a straight-chain alkylene group or a branched-chain alkylene group, for example, C 1-20 Alkylene, preferably C 1-10 Alkylene, more preferably C 1-6 Alkylene or C 1-4 Alkylene, more preferably C 1-2 Alkylene. In addition, as the halogenated alkyl group, the carbon atom to which the halogenated group is bonded is preferably directly connected to the carbonyl group or aromatic ring contained in B. In addition to being bonded to a mercapto group, the halogenated alkyl group may also be bonded to an amino group.

[0126] The activated carbonyl group includes activated ester group, formyl group and the like. Examples of activated ester groups include imide ester groups such as succinimidyl groups, 4-nitrophenol ester groups, HOBt ester groups, HOAt ester groups, and Oxyma ester groups. The activated carbonyl group can be bonded to an amino group in addition to the side chain thiol such as a cysteine ​​residue. In addition, the formyl group can be bonded to the side chain amino group of lysine by, for example, a reductive amination reaction.

[0127] The unsaturated hydrocarbon group refers to an unsaturated hydrocarbon group having at least one carbon-carbon double bond or carbon-carbon triple bond, including vinyl, propargyl, etc., preferably vinylcarbonyl, propargylcarbonyl, vinylsulfonyl, and the like. The unsaturated hydrocarbon group can be bonded to an amino group or a mercapto group by, for example, Michael addition or nucleophilic substitution reaction.

[0128] Examples of sulfonyl-containing groups include alkylsulfonyl, arylsulfonyl, and sulfonate groups (for example, alkylsulfonyloxy, arylsulfonyloxy, etc.). The leaving group can react with sulfhydryl and amino groups. Examples of the alkylsulfonyl group include methanesulfonyl, chloromethanesulfonyl, and trifluoromethanesulfonyl. Examples of the arylsulfonyl group include benzenesulfonyl and tosylsulfonyl. In addition, examples of the sulfonate group include a methanesulfonyloxy group, a chloromethanesulfonyloxy group, a trifluoromethanesulfonyloxy group, a benzenesulfonyloxy group, and a tosyloxy group.

[0129] As the carbene-forming group, for example, a diazonium-containing group, a Diazirinyl structure-containing group, etc. can be cited, and preferably, a group in which a diazonium group is bonded to a carbon atom adjacent to a carbonyl group is mentioned. Since the diazo group is detached, carbene can be generated and bonded to thiol. In addition, as a carbene-containing group, a group obtained by generating carbene from various carbene-generating groups can be cited.

[0130] Disulfide bond-containing groups and sulfhydryl groups can form disulfide bonds with the side chain sulfhydryl groups of cysteine ​​residues.

[0131] Epoxy groups, isocyanate groups, and thioisocyanate groups can react with both mercapto groups and amino groups.

[0132] The number of A is preferably an integer of 2 or more as a. If A is 2 or more and the side chain reactive functional group is 2 or more, the polypeptide chain can be cyclized. The cyclized polypeptide chain can further improve stability and so on.

[0133] In the above formula, B represents a linking group or a single bond. Examples of B as the linking group include heteroatom-containing polar groups, aliphatic hydrocarbon groups, and aromatic rings.

[0134] Examples of polar groups containing heteroatoms include: -O-, -S-, -NR 1 -(In the formula, R 1 Represents hydrogen atom, hydrocarbon group (preferably C 1-6 Alkyl) or the atom bond at the end of the linking group. It should be noted that when R is an atomic bond, the valence of the linking group is 3 (the same applies below)), -CO-, -COO-, -CONR 2 -(In the formula, R 2 Is a hydrogen atom, a hydrocarbon group (preferably C 1-6 Alkyl) or the atom bond at the end of the linking group), -C (=N-R 3 )-(Where, R 3 It means that it includes a chain or cyclic aliphatic hydrocarbon group that may have a substituent, an aromatic cyclic group that may have a substituent, or a heteroatom-containing group, and a functional group for adding a certain function At least one or more groups in the composition group, hydrogen atom or atom bond at the end of the linking group), -N=N-, and -SO 2 -.

[0135] The aliphatic hydrocarbon group may be any one of linear alkylene, branched alkylene, and cyclic alkylene, for example, C 1-20 Alkylene, preferably C 1-10 Alkylene, more preferably C 1-6 Alkylene or C 1-4 Alkylene, more preferably C 2-4 Alkylene. The above-mentioned heteroatom-containing polar group may be inserted between the carbon atoms or the terminal of the aliphatic hydrocarbon group, and it may have a substituent. As a substituent, a halogeno group, an aryl group, a carboxyl group, an alkoxycarbonyl group, or a hydroxyl group can be mentioned. As the aryl group, C is preferred 6-10 Aryl, more preferably phenyl or naphthyl, preferably phenyl. The number of substituents is not particularly limited as long as it can be substituted. For example, it may be 1 or more and 4 or less, preferably 3 or less or 2 or less, and preferably one. When the number of substituents is 2 or more, these substituents may be the same or different from each other.

[0136] As the aromatic ring group, phenyl, indenyl, naphthyl, biphenyl and the like are preferred. 6-10 Aryl, more preferably phenyl or naphthyl, preferably phenyl. The aromatic ring group may have a substituent. Examples of the substituent include a halogenated group, an alkyl group, and preferably C 1-6 Alkyl, aralkyl, preferably benzyl, carboxy, alkoxycarbonyl, preferably (C 1-6 Alkoxy) carbonyl, hydroxyalkyl, preferably hydroxy-C 1-6 Alkyl or carboxyalkyl, preferably carboxy-C 1-6 alkyl. The number of substituents is not particularly limited as long as it can be substituted. For example, it may be 1 or more and 4 or less, preferably 3 or less or 2 or less, and preferably one. When the number of substituents is 2 or more, these substituents may be the same or different from each other.

[0137] The aforementioned linking group B preferably has -B1-unit, -B2-unit, -B2-B1-unit or -B2-B1-B3-unit. B1 is a chain or cyclic aliphatic hydrocarbon group selected from the group consisting of a heteroatom-containing polar group optionally inserted between carbon atoms and optionally having substituents, alone or in combination, and an aromatic group optionally having substituents One or more groups in the group ring, B2 and B3 are each independently a heteroatom-containing polar group, and B1 or B2 is bonded to the aforementioned A. Here, the heteroatom-containing polar group and the substituent have the same meaning as described above.

[0138] Examples of groups containing heteroatoms include: -O-, -S-, -NR 3 -(In the formula, R 3 Represents hydrogen atom, hydrocarbon group (preferably C 1-6 Alkyl) or the atom bond at the end of the linking group. It should be noted that when R is an atomic bond, the valence of the linking group is 3 (the same applies below)), -CO-, -COO-, -CONR 4 -(In the formula, R 4 Is a hydrogen atom, a hydrocarbon group (preferably C 1-6 Alkyl group) or the atom bond at the end of the linking group), -N=N-, and -SO 2 -.

[0139] In the above formula, C represents a functional group for adding a certain function to the polypeptide. The functional group may be appropriately selected according to the purpose of use, and is not particularly limited. For example, in addition to the linker compound that cyclizes the polypeptide, luminescent substances such as fluorescent substances, pigments, radioactive substances, pharmaceuticals, toxins, Nucleic acids, amino acids, peptides, carbohydrates, lipids, various polymers, etc. and their combinations. Examples of fluorescent substances include fluorescent dyes of fluorescein, rhodamine, coumarin, pyrene, and cyanine.

[0140] As a specific modification reagent, for example, any one represented by the following formula can be cited.

[0141]

[0142] (In the formula, A, B1, B2, and B3 are the same as the above. n represents an integer of 1 or more. Any one or more of B1, B2, and B3 may optionally be bonded to one or more of the foregoing C)

[0143] It should be noted that the compounds represented by the aforementioned formulas (1a) to (1f) have a functional group C (a group containing any one of a fluorescent dye and a labeling unit), and the functional group C and B1, B2, Any one of B3 is bonded. In addition, in order to improve water solubility, optional sulfonic acid groups (-SO 2 -OH), sulfonate group (-SO 2 -O-M + ) And other water-soluble substituents. As M + Examples include alkali metal ions such as sodium ion and potassium ion.

[0144] Specific examples of formula (1a) include, for example: A is a halogenated alkyl group, an activated carbonyl group (especially an activated ester group) or an epoxy group, B1 is an aliphatic hydrocarbon group or an aromatic ring, and n is a compound of 2 or 3; especially The compound represented by the following formula is included.

[0145]

[0146] The specific examples of formula (1b) include: A is a halogenated alkyl group, B2 is a polar group containing an oxygen atom (especially -CO-) or a polar group containing a nitrogen atom (especially -C(=N-R) 5 )-(Where, R 5 It means that it includes a chain or cyclic aliphatic hydrocarbon group that may have a substituent, an aromatic cyclic group that may have a substituent, or a heteroatom-containing group, and a functional group for adding a certain function In the composition group, at least one or more groups, a hydrogen atom, or an atomic bond at the end of the linking group), a compound where n is 2; in particular, a compound represented by the following formula is included.

[0147]

[0148] Specific examples of formula (1c) include: A is a halogenated alkyl group or an activated carbonyl group (especially an activated ester group), B1 is an aliphatic hydrocarbon group, an aliphatic hydrocarbon group or an aromatic ring with an oxygen atom inserted between carbon atoms, and B2 It is a polar group containing an oxygen atom, a nitrogen atom, etc. (especially -COO-, -CONH-), and a compound in which n is 1, 2 or 3; in particular, it includes a compound represented by the following formula.

[0149]

[0150] Specific examples of formula (1d) include: A is a halogenated alkyl group, B1 is an aliphatic hydrocarbon group, B2 is a polar group containing oxygen and nitrogen atoms (especially -CONH-), and n is a compound of 3; especially The compound represented by the following formula.

[0151]

[0152] Specific examples of formula (1e) include: A is a haloalkyl group, B1 is an aromatic ring, B2 and B3 are polar groups containing oxygen atoms, nitrogen atoms, etc. (especially -COO-, -N=N-, etc.) ), a compound where n is 2; in particular, it includes a compound represented by the following formula.

[0153]

[0154] The conditions for modifying the polypeptide chain in the RD complex can be appropriately set according to the type of side chain reactive functional group to be modified and the modification reagent used. For example, when it is necessary to modify the side chain sulfhydryl group of a cysteine ​​residue, the disulfide bond is cleaved by a reducing agent to form a sulfhydryl group and then reacted with a modifying reagent. Examples of the reducing agent include tris(2-carboxyethyl)phosphine sodium salt, dithiothreitol, β-mercaptoethanol, and the like.

[0155] In addition, when reacting the halogenated alkyl group, epoxy group, etc. of the modifying reagent with the side chain amino group, a base may be optionally added. As the base, for example, bicarbonate such as sodium bicarbonate; carbonate such as sodium carbonate; metal hydroxide such as sodium hydroxide; organic base such as pyridine and triethylamine, and the like.

[0156] As a reaction solvent when reacting the modifying reagent, water can usually be used. The reaction temperature is, for example, about 0 to 30°C, preferably about 1 to 20°C, and more preferably about 1 to 10°C.

[0157] The pH during the reaction of the modifying reagent may be appropriately adjusted according to the modifying reagent used, and is not particularly limited. For example, it can be selected from the range of about 4.0 to 10.0, preferably about 5.0 to 9.0, and more preferably about 6.0 to 8.0. The more preferable range varies depending on the modification reagent, but from the viewpoint of inhibiting the introduction of two or more modification reagents per polypeptide chain, the pH can be adjusted to 7.0 to 7.5.

[0158] The amount of the modification reagent can be appropriately set according to the type of reagent. For example, relative to 1 mol of the ribosome complex containing the unmodified polypeptide chain, it is, for example, 1,000 mol or more, preferably 10,000 mol or more, and more preferably 60,000 mol or more. More preferably, it is 100,000 mol or more. The upper limit is not particularly limited. For example, it is 100,000,000 mol or less, preferably 50,000,000 mol or less, more preferably 20,000,000 mol or less, and still more preferably 10,000,000 mol or less.

[0159] After modifying the polypeptide chain, the RD complex can be purified by conventional methods. For example, when there are tag sequences such as FLAG (registered trademark) sequence and poly-His sequence in the polypeptide chain, a known purification method corresponding to these sequences can be used. It should be noted that the carrier to which an antibody specific to the tag sequence is bound can be bound to the RD complex with affinity before reacting the modification reagent. The affinity binding is not cut off during the reaction of the modification reagent, and the affinity binding can be used to purify the RD complex after the reaction of the modification reagent.

[0160] The RD complex produced by the method of the present invention described above includes a polypeptide chain, an mRNA molecule and a ribosome. The polypeptide chain is selected from cysteine ​​residues, lysine residues, histidine residues and tryptophan. One or more reactive amino acid residues in the residue group are modified by the modification reagent used, and the mRNA molecule includes a base sequence encoding the amino acid sequence of the polypeptide chain. In the RD complex having the aforementioned formulas (1), (1a) to (1e), the group A is replaced with the group Ax as a result of the reaction (Ax is a cysteine ​​residue, a lysine residue From this point of view, the structure of the bonding group formed by the reaction of the side chain of the group, histidine residue or tryptophan residue can be distinguished from the unmodified RD complex.

[0161] This application claims priority based on Japanese Patent Application No. 2016-113935 filed on June 7, 2016. The entire content of the specification of Japanese Patent Application No. 2016-113935 filed on June 7, 2016 is incorporated into this application by reference.

[0164] Example 1

[0165] (1) Preparation of RNA library

[0166] In this item (1), the NNK method contains 10 12 The method of the RNA library of the above RNA is described, the RNA has the (NNK) 10 [In the formula, N represents A, U, G or C, K represents G or U, and NNK corresponds to all codons] the sequence of the sequence.

[0167] To make the RNA library, use figure 1 The structure of the template DNA (base sequence: sequence number 1, amino acid sequence: sequence number 2). Specifically, the reaction solution having the composition shown in Table 1 was used, and the plasmid was used as the template DNA in the PCR cycle of Table 2 to prepare a 5'fragment. In Table 1, 5FFnew_130816 is the forward primer, and Ma3frag_R0502 is the reverse primer.

[0168] [Table 1]

[0169]

[0170] [Table 2]

[0171]

[0172] Next, using the reaction solution having the composition shown in Table 3, a 3'fragment of template DNA was prepared in accordance with the PCR cycle in Table 4. In Table 3, Ma10NNK_F0502 is a forward primer and 3F-R is a reverse primer.

[0173] [table 3]

[0174]

[0175] [Table 4]

[0176]

[0177] Next, using the reaction solution having the composition shown in Table 5, overlap PCR was performed according to the PCR cycle of Table 6, and the 5'fragment and the 3'fragment were ligated to amplify the full length to obtain template DNA. It should be noted that in Table 5, X~Z means using 1×10 12 The 5’ and 3’ fragments of, add H to the reaction solution 2 O and adjust the total amount to 60 μL.

[0178] [table 5]

[0179]

[0180] [Table 6]

[0181]

[0182] By using the obtained template DNA as a template, using a reaction solution having the composition shown in Table 7, and reacting at 37°C for 5 hours, 10 containing the base sequence of SEQ ID NO: 3 was obtained. 12 RNA library of the above mRNA. The mRNA contained in the library is as figure 1 Shown are FLAG (registered trademark) site, His6 site, random sequence, TEV protease site, spacer sequence in order from the 5'side, and no stop codon.

[0183] [Table 7]

[0184]

[0185] (2) Preparation of ribosome display complex library

[0186] Using a recombinant cell-free protein synthesis kit ("PURE frex (registered trademark)" manufactured by GeneFrontier Corporation), a ribosome display (RD) complex was prepared based on the aforementioned RNA library. Separately streptavidin-magnetic particles ("NanoLink TM 5 μL of Streptavidin MagneticBeads "Solulink Co., Ltd.) was diluted to 150 μL. The RD complex reaction solution and anti-FLAG (registered trademark) M2 antibody-conjugated agarose beads (manufactured by Sigma-Aldrich Co., 20 μL) were mixed and stirred at 4°C for 60 minutes. The anti-FLAG M2 antibody-bound sepharose beads in which the RD complex with the FLAG sequence was selectively bound to the peptide portion were recovered.

[0187] (3) Cyclization reaction of peptide

[0188] After diluting the agarose beads recovered above to 80μL, add 10mM tris(2-carboxyethyl)phosphine hydrochloride (4μL) as a reducing agent (final concentration 0.5mM) and 40mM 1,3-bis as a modification reagent Bromo-2-acetone (4μL) (final concentration 2mM), cyclization reaction was carried out at 4°C for 3 hours. It should be noted that the addition of reducing agent is not necessary at this time. After the cyclization reaction, the RD complex was separated from the agarose beads by adding FLAG peptide.

[0189] (4) Selection of HSP90 affinity peptide

[0190] Separately, the above-mentioned streptavidin-magnetic particle diluent (5μL) was mixed at a molar ratio of 1:1 to combine biotin with HSP90 as a heat shock protein, and stirred at 4°C to make HSP90 Combined with magnetic particles. In addition, a magnetic particle suspension was prepared in the same manner except that HSP90 was not bound for comparison.

[0191] The peptide cyclized RD complex solution obtained in (3) above and the magnetic particle suspension were mixed, and stirred at 4°C for 1 hour. A magnetic stand was used to recover the magnetic particles, and 0.05 M EDTA was added, thereby separating RNA from the RD complex bound to HSP90 on the magnetic particles. After removing the magnetic particles using a magnetic stand, RNA was purified using an RNA concentration/purification kit ("RNeasy MinElute Cleanup Kit" manufactured by QIAGEN). Next, after reacting with the composition shown in Table 8 at 65°C for 5 minutes, the reaction liquid was reacted with the composition shown in Table 9 at 50°C for 1 hour, and further at 70°C for 15 minutes. Reverse transcription.

[0192] [Table 8]

[0193]

[0194] [Table 9]

[0195]

[0196] The cDNA obtained in the above reverse transcription reaction was used in RT-PCR to amplify a sequence containing a random sequence. Specifically, using the reaction solution of the composition shown in Table 10, PCR was performed through the PCR cycle of Table 11.

[0197] [Table 10]

[0198]

[0199] [Table 11]

[0200]

[0201] In addition, in (1) above, the heating time at 68°C in the PCR cycle was changed to 15 seconds, except that the 5'fragment (SEQ ID NO: 6) was similarly prepared, and 3fragF_140407, 3fragF_140407, In addition, the heating time at 68°C in the PCR cycle was changed to 25 seconds, and a 3'fragment (SEQ ID NO: 7) was prepared in the same manner. Next, in addition to using the obtained 5'fragment and 3'fragment, a cDNA solution containing the above-mentioned random sequence was used, and the amount of polymerase was changed to 0.6 μL, and the same procedure was performed except that the fragments were ligated by overlapping PCR and amplified Increase the DNA obtained.

[0202] Using the obtained DNA, the mRNA was transcribed in the same manner as in (1) above, and the step (2) "Preparation of ribosome display complex library" was returned to the above step. Below, the RD complex with affinity for HSP90 was recovered in the same manner as above. . Repeat the above operation until it is confirmed that the amount of mRNA contained in the RD complex bound to HSP90 no longer increases. Hereinafter, one repetition is referred to as "round". In the first to third rounds, the molar ratio of the RD complex to the used HSP90 was adjusted to RD complex: HSP90=3:1, and in the fourth to fifth rounds, the molar ratio was adjusted to 10:1. The results are shown together with the amount of mRNA before contact with HSP90 and the amount of mRNA when HSP90 is not used figure 2. Such as figure 2 As shown in the results, the RD complex bound to HSP90 increased in the second round compared to the first round, but no increase was observed after the second round, and it is considered that the RD complex with affinity for HSP90 was completed Of concentration.

[0203] (5) Identification of HSP90 affinity peptide

[0204] Using the full-length DNA of the round whose sequence concentration was confirmed as a template, PCR was performed using Taq polymerase, thereby adding A to the end. Using this overhang and cloning kit ("pGEM T Easy Cloning Kit" manufactured by Promega), it was ligated to the attached plasmid DNA. The obtained plasmid was used to transform and culture JM109 competent cells. The amino acid sequence of the above-mentioned full-length strand DNA was analyzed using plasmids of each clone extracted from the colonies formed by the culture. Table 12 shows the sequence of the random part (NNK part) in the obtained amino acid sequence.

[0205] [Table 12]

[0206]

[0207] As shown in Table 12, the screening method of the present invention can be used from 10 12 16 peptides encoding peptides with affinity to HSP90 were selected from the above mRNA library. In addition, it was confirmed that the amino acid sequences of the selected peptides were similar.

[0208] (6) Confirmation of the binding ability of the obtained sequence

[0209] The affinity of each clone obtained in (5) above to HSP90 was confirmed. Specifically, using the full-length DNA of each clone synthesized by the above (5) as a template, RNA was synthesized in the same manner as in the above (1), and a ribosome display complex was prepared in the same manner as in the above (2). In addition, a part of the polypeptide was cyclized in the same manner as in (3) above. The affinity of each obtained ribosome display complex with HSP90 was investigated in the same manner as in (4) above. By quantifying the amount of recovered complexes, the affinity of each clone to HSP90 was determined. Show the result in image 3. image 3 The letters in are equivalent to the sequence symbols in Table 12 above.

[0210] Such as image 3 The results shown show that among the ribosome display complexes in which the affinity with HSP90 was studied, three polypeptides showed affinity for HSP90, and for two of them, it was suggested that the loops connected to the modification reagent Bonding is enhanced by more than 10 times in the state. These results indicate that the polypeptide presented on the RD complex can be linked to the modification reagent while maintaining the function of the RD complex. In addition, depending on the presence or absence of a modification reagent, a clone with an affinity of about 6 times can be obtained. That is, it was suggested that the affinity with HSP90 was increased by cyclizing the polypeptide, and the usefulness of the linking modification reagent was shown.

[0211] Example 2: Cyclization reaction of peptide

[0212] Using a recombinant cell-free protein synthesis kit ("PURE frex (registered trademark)" manufactured by GeneFrontier Corporation), 50 μL of the reaction solution was mixed with RNA having FLAG sequence, His6 sequence, and base sequence encoding TEV protease site (serial number 8) 2.5×10 13 The molecules were reacted at 37°C for 35 minutes to prepare an RD complex. Anti-FLAG (registered trademark) M2 antibody-conjugated agarose beads (manufactured by Sigma-Aldrich, 2 μL) were added to the reaction solution and bound to the RD complex. Furthermore, tris(2-carboxyethyl) sodium salt (pH7, final concentration 0.5mM) and Figure 4 Each modification reagent at the indicated final concentration of 2 mM was stirred at 4°C for 3 hours to cyclize the peptides in the RD complex on the beads. After the reaction, the RD complex was released from the beads by adding FLAG peptide (sequence: DYKDDDDK, 5 mg). Separate and remove beads from the reaction solution, and add no Mg 2+ After dissociating the complex with phosphate buffered saline (pH 7.5, 100 μL), the peptide chain was purified by His-tag beads. After the purified polypeptide was cleaved with TEV protease, the molecular weight of the fragment peptide (SEQ ID NO: 9) containing the cyclization site and the N-terminal formylation was measured by MALDI-TOFMS. The chemical structure of the modified reagent and the mass spectrum obtained are shown in Figure 4. Figure 4 in, Figure 4 (A) is the mass spectrum of the reaction solution without modification reagents, Figure 4 (E) is a mass spectrum of a reaction solution that has been reacted in the same way except that RNA is not used, and a signal showing the background. In addition, white arrows indicate peaks of uncyclized peptide chains, and black arrows indicate peaks of cyclized peptide chains.

[0213] Such as Figure 4 As shown in the results shown, it can be seen that the case (b) when 1,3-dibromo-2-propanone is used as the modification reagent, and the case (c) when 1,3-bis(bromomethyl)benzene is used for cyclization 化反应。 Chemical reaction. In the case of using 1,5-hexaazabenzene diepoxide as the modifying reagent (d), the reaction efficiency is relatively low but it can be confirmed as a cyclic compound, so it is considered that the epoxide can also be used as a cyclization reagent.

[0214] It should be noted that, in each mass spectrum, a peak in which the used modification reagent increased the molecular weight of one molecule was clearly observed, on the other hand, a peak in which the molecular weight of two or more molecules increased was not clearly observed. Based on the above results, it can be considered that only one molecule of the modification reagent is bonded to the polypeptide chain.