Method for preparing polypeptide solution for evaluating physiological activity and method for evaluating physiological activity of polypeptide

Abstract

The present invention provides a method for preparing a polypeptide solution for evaluating physiological activity and a method for evaluating the physiological activity of a polypeptide, the method comprising the steps of: (A) bringing a crude polypeptide solution after cell-free expression into contact with a carrier so as to attach a polypeptide having a pH-responsive affinity tag to the carrier; (B) eluting the polypeptide attached to the carrier with less than 50 mM of an acidic aqueous solution or an alkaline aqueous solution to obtain an eluate; and (C) adding an aqueous alkaline solution or an aqueous acidic solution to the eluate in order to neutralize the acid or alkali contained in the eluate, and neutralizing the eluate so that the salt concentration formed in the neutralization reaction is less than 50 mM, thereby obtaining a polypeptide solution.

Description

Technical Field

[0001] This invention relates to a method for preparing a polypeptide solution for evaluating physiological activity and a method for evaluating the physiological activity of polypeptides. Background Technology

[0002] As compounds that combine the low manufacturing cost and high stability of low-molecular-weight compounds with the protein-binding properties of high-molecular-weight compounds such as antibodies, medium-molecular-weight compounds, especially peptides, are attracting attention and beginning to be used in a wide range of bio-related industries, primarily therapeutic and diagnostic drugs. Among these, there is active development of specialized peptides that achieve high target protein binding and stability by introducing non-natural amino acids not found in natural sources.

[0003] In recent years, special peptides that not only bind to proteins but also exhibit physiological activities such as cell proliferation through protein binding have begun to attract attention as previously undiscovered high-functionality compounds. However, the chemical synthesis of these special peptides is costly and time-consuming, and the special peptides exhibiting physiological activity are only a very small fraction of protein-binding special peptides, making their search extremely difficult.

[0004] As a method for searching for peptides exhibiting physiological activity against cells, Patent Document 1 reports a method for rapidly and simply searching for physiological activity against cells by diluting a peptide synthesized using a rapid and low-cost cell-free expression method.

[0005] Methods for purifying polypeptides synthesized using cell-free expression methods for targeted applications are also known. Non-Patent Literature 1 describes affinity purification of polypeptides synthesized using cell-free expression. Existing technical documents Patent documents

[0006] Patent Document 1: International Publication No. 2022 / 158554 Non-patent literature

[0007] Non-Patent Literature 1: Purification Method for His-Tagged Proteins, [Online], 2021, GeneFrontier Corporation, [Searched March 20, 2023], Internet (URL: https: / / www.genefrontier.com / cases / purefrex / applications-09 / ) Summary of the Invention The technical problem to be solved by the invention

[0008] When synthesizing special peptides containing non-natural amino acids using cell-free expression methods, the resulting peptide concentrations are extremely low. Therefore, as described in Patent Document 1, it is difficult to ensure a sufficient concentration for activity evaluation when diluting after cell-free expression. On the other hand, when using the cell-free expressed peptide solution without dilution for evaluating cellular physiological activity, cell death due to the cytotoxicity of components contained in the solution makes it difficult to assess physiological activity. Therefore, the method in Patent Document 1 is not suitable for special peptides containing non-natural amino acids.

[0009] To remove toxic components from the peptide solution after cell-free expression, affinity purification, as described in Non-Patent Literature 1, can be considered. However, in common affinity purification methods, the eluent is cytotoxic due to high concentrations of imidazole doping, high concentrations of eluent peptide doping, and low pH, making it unsuitable for direct evaluation of cellular physiological activity. Performing additional steps to remove dopants results in peptide loss and is also costly.

[0010] In view of this situation, the present invention relates to a method for preparing a peptide solution for evaluating physiological activity and a method for evaluating the physiological activity of peptides synthesized by cell-free expression, which can be easily evaluated at high peptide concentrations. means for solving technical problems

[0011] The methods used to solve the above problems include the following approaches. <1> A method for preparing a polypeptide solution for evaluating physiological activity, wherein the method includes the following steps: (A) The crude solution of the cell-free expressed peptide is contacted with the carrier so that the peptide with a pH-responsive affinity tag attaches to the carrier; (B) The polypeptide attached to the carrier is eluted with an acidic or alkaline aqueous solution of less than 50 mM to obtain an eluent; and (C) To neutralize the acid or alkali contained in the eluent, an alkaline or acidic aqueous solution is added to the eluent, and the neutralization is carried out in such a way that the salt concentration formed in the neutralization reaction is less than 50 mM to obtain a polypeptide solution. <2> A method for evaluating the physiological activity of a polypeptide, wherein the method includes the following steps: Prepare polypeptide solutions using the method described in <1>; and To evaluate the physiological activity of peptides, the peptide solution is brought into contact with cells. <3> According to the method described in <1> or <2>, wherein, Peptides contain non-natural amino acid residues. <4> The method according to any one of <1> to <3>, wherein, A polypeptide contains one or more ring structures. <5> According to the method described in <4>, wherein, The ring structure includes the following structure. [Chemical Formula 1] In the formula, X 1 and X 2 Each can be an independent linking group, and n represents an integer from 1 to 10. <6> According to the method described in <4> or <5>, wherein, Polypeptides contain 1 to 4 ring structures. <7> The method according to any one of <1> to <6>, wherein, In step (B), an acidic aqueous solution of less than 50 mM is used, and in step (C), an alkaline aqueous solution is used. <8> According to the method described in <7>, wherein, The acidic aqueous solution is hydrochloric acid solution. <9> According to the method described in <7> or <8>, wherein, The alkaline aqueous solution is an aqueous solution of sodium bicarbonate, an aqueous solution of sodium carbonate, or a mixed aqueous solution of sodium bicarbonate and sodium carbonate. <10> The method according to any one of <1> to <9>, wherein, The carrier is a magnetic bead. <11> The method according to any one of <1> to <10>, wherein, Affinity tags include FLAG, HA, His, c-Myc, V5, Strep, or PA tags. <12> The method according to any one of <1> to <11>, wherein, The method further includes a cell-free peptide expression step before (A). <13> The method according to any one of <1> to <12>, wherein, The method, following (A) and preceding (B), further includes a step of washing the carrier with water. Invention Effects

[0012] According to the present invention, a method for preparing a peptide solution for evaluating physiological activity and a method for evaluating the physiological activity of peptides synthesized by cell-free expression can be provided, which can easily evaluate the physiological activity of peptides synthesized by cell-free expression at high peptide concentrations. Detailed Implementation

[0013] The embodiments of the present invention will be described below. These descriptions and examples are illustrative and not intended to limit the scope of the embodiments. The mechanisms of action described in the present invention include speculation, and their correctness does not limit the scope of the embodiments.

[0014] In this invention, the term "process" or the term representing a process includes not only processes that are independent of other processes, but also processes that can achieve their purpose, even if they cannot be clearly distinguished from other processes. In this invention, the numerical range represented by “~” indicates the range encompassed by the numerical values ​​recorded before and after “~” as the minimum and maximum values, respectively. Within the numerical ranges described in stages in this invention, the upper or lower limit value described in one numerical range can be replaced with the upper or lower limit value of other numerical ranges described in stages. Furthermore, within the numerical ranges described in this invention, the upper or lower limit value of the numerical range can be replaced with the values ​​shown in the embodiments. In this invention, a variety of substances corresponding to each component may be included. In this invention, when referring to the amount of each component in the composition, unless otherwise specified, the amount refers to the total amount of the various substances present in the composition, in cases where multiple substances corresponding to each component are present in the composition. In this invention, amino acids are labeled using the 3-character and 1-character labels specified by IUPAC-IUBMB JCBN (IUPAC-IUBMB Joint Commission on Biochemical Nomenclature). Unless otherwise specified, the amino acids mentioned in this invention are L-amino acids. In this invention, the unit "M" is the unit of molar concentration, which has the same meaning as mol / L. In this invention, even when the elements are expressed in a singular form, unless otherwise expressly stated, the existence of multiple elements is not excluded as long as it does not create a technical contradiction.

[0015] <Methods for preparing peptide solutions for evaluating physiological activity and methods for evaluating the physiological activity of peptides> A method for preparing a polypeptide solution for evaluating physiological activity according to one embodiment of the present invention includes the following steps: (A) The crude solution of the cell-free expressed peptide is contacted with the carrier so that the peptide with a pH-responsive affinity tag attaches to the carrier; (B) The polypeptide attached to the carrier is eluted with an acidic or alkaline aqueous solution of less than 50 mM to obtain an eluent; and (C) To neutralize the acid or alkali contained in the eluent, an alkaline or acidic aqueous solution is added to the eluent, and the neutralization is carried out in such a way that the salt concentration formed in the neutralization reaction is less than 50 mM to obtain a polypeptide solution. An embodiment of the present invention provides a method for evaluating the physiological activity of a polypeptide, comprising the following steps: preparing a polypeptide solution by the method described above for preparing a polypeptide solution for evaluating physiological activity; and (D) contacting the polypeptide solution with cells to evaluate the physiological activity of the polypeptide.

[0016] In the method for preparing peptide solutions and evaluating physiological activity of the present invention (hereinafter, both are collectively referred to as "the method of the present invention"), affinity purification is used to purify the peptide after cell-free expression. Affinity purification refers to a purification method in which the molecule to be purified is specifically attached to a carrier in a non-covalent manner, followed by elution from the monomer. According to the method of the present invention, the physiological activity of the peptide can be evaluated simply and at a high peptide concentration. In the method of the present invention, the peptide attached to the carrier is eluted with an acidic or alkaline aqueous solution of less than 50 mM, and then the eluent is neutralized with a salt concentration of less than 50 mM. The resulting peptide solution has a sufficiently low salt concentration, so physiological activity can be evaluated under conditions with minimal influence of osmotic pressure, and it contains almost no components other than salt. Furthermore, since the cytotoxic components contained in the peptide solution after cell-free expression are sufficiently reduced, the evaluation of physiological activity can be performed directly on cells while reducing the influence of cytotoxicity. Thus, peptide solutions can be prepared rapidly and at low cost, and physiological activity can be evaluated.

[0017] Hereinafter, the processes (A) to (D) mentioned above will also be referred to as processes (A) to (D). Furthermore, process (A) is sometimes referred to as the "attachment process", process (B) as the "washing and extraction process", process (C) as the "neutralization process", and process (D) as the "evaluation process". In addition to steps (A) to (D), the method of the present invention may include other steps. For example, the method of the present invention may include a step of washing the carrier with water after step (A) and before step (B). Furthermore, the method of the present invention may include a step of expressing the polypeptide without cells before step (A). The following describes each step in the method of the present invention.

[0018] [No cell expression] The method of the present invention may include a cell-free polypeptide expression step prior to (A). “Cell-free expression” refers to a method of expressing polypeptides from template nucleic acids in vitro without using cells (such as E. coli), but using components derived from living cells or obtained through genetic engineering methods (ribosomes, transcription / translation factors, etc.).

[0019] Compared to expression using cells such as E. coli, cell-free expression offers the following advantages: First, it is highly operable and offers greater system flexibility because it does not require maintaining living cells. Therefore, it is possible to design synthetic systems with various modifications based on the properties of the target peptide. Second, while it is virtually impossible to synthesize peptides toxic to the cells used in cell-based expression systems, cytotoxic peptides can be synthesized in cell-free expression systems. Third, high-throughput synthesis is easily achieved because multiple peptides can be synthesized simultaneously and rapidly, and the resulting peptides are easy to isolate and purify. Fourth, it allows for the synthesis of specialized peptides containing non-natural amino acids.

[0020] -peptide- Cell-free expressed polypeptides are polypeptides to which a pH-responsive affinity tag is attached as an evaluation target for physiological activity (hereinafter also referred to as "evaluation target polypeptide"). In this invention, a polypeptide refers to a molecule composed of amino acids linked by peptide bonds. There is no limitation on the number of amino acid residues in a polypeptide, and polypeptide is a term encompassing proteins.

[0021] The evaluated polypeptide is any polypeptide whose physiological activity on cells is to be evaluated. In this invention, the "physiological activity" of a polypeptide refers to its physiological activity on living cells. Examples of physiological activity include activity on cell proliferation, cell differentiation state, cell viability, signaling of various intracellular proteins, and responses to various reporter genes. In the evaluation process, the evaluated polypeptide is brought into contact with cells, and changes in the state of the cells are observed, thereby enabling the evaluation of the physiological activity of the evaluated polypeptide.

[0022] From the viewpoint of peptide activity, the number of amino acid residues in the target peptide is preferably 6 or more. From the viewpoint of expression efficiency, the number of amino acid residues in the target peptide is preferably less than 600, more preferably less than 300, and even more preferably less than 150. From this viewpoint, the number of amino acid residues in the target peptide is preferably 6 or more and less than 600, more preferably 6 or more and less than 300, and even more preferably 6 or more and less than 150.

[0023] pH-responsive affinity tags are tags that demonstrate the property of the molecule to be purified to attach to a carrier at a specific pH and to dissociate from the carrier at a specific pH. From the viewpoint of peptide stability, affinity tags that attach to the carrier at neutral conditions (e.g., pH 6–8) and dissociate from the carrier at acidic conditions (e.g., pH 4 or below) are preferred. Affinity tags can be used as long as they exhibit pH responsiveness, but from the viewpoint of minimizing their impact on the physiological activity of the target peptide, tags consisting of a small number of amino acids are preferred. Examples of tags with a small number of amino acids include FLAG tags, HA tags, His tags, c-Myc tags, V5 tags, Strep tags, and PA tags. Among these, from the viewpoint of purification yield, HA tags, His tags, c-Myc tags, and PA tags are more preferred.

[0024] When expressing peptides in cell-free environments, spacer sequences may be inserted at the N-terminus, C-terminus, and / or within the peptide sequence, or not. From the viewpoint of purification yield, it is preferable to insert spacer sequences before or after the affinity tag. Furthermore, from the viewpoint of minimizing impact on the physiological activity of the target peptide, it is preferable to insert a spacer sequence between the target peptide and the affinity tag. GGS linkers can be cited as examples of spacer sequences.

[0025] In one approach, the expressed polypeptide preferably contains a translation-promoting sequence in its N-terminal translation region. By including a translation-promoting sequence in the polypeptide, the cell-free expression level of the base sequence encoding the polypeptide is increased. As the translation-promoting sequence, a base sequence encoding the following amino acid sequence (1) is preferred. Hereinafter, the polypeptide having the amino acid sequence (1) will also be referred to as the "VKKX tag".

[0026] Amino acid sequence (1): Val-Lys-Lys-(Xaa) n In the amino acid sequence (1), (Xaa) n Let n be any n amino acids linked together, where n is an integer from 1 to 8, forming (Xaa). n The amino acid can be one type or two or more types.

[0027] As a component (Xaa) n The amino acids are preferably selected from the group consisting of Ile, Lys, Arg, His, Ser, Thr, Asp, Cys, Asn, Tyr, Gln, Trp and Phe, and more preferably selected from the group consisting of Ile, Lys, Arg, His, Ser, Thr and Asp. In the amino acid sequence (1), n ​​is preferably an integer from 1 to 7, and more preferably an integer from 2 to 7.

[0028] As a VKKX tag, a polypeptide having an amino acid sequence (2) is preferred, a polypeptide having an amino acid sequence (3) is more preferred, and a polypeptide having an amino acid sequence (4) is even more preferred.

[0029] Amino acid sequence (2): Val-Lys-Lys-Xaa-(Xaa) m In the amino acid sequence (2), the fourth Xaa from the N-terminus is a single amino acid selected from the group consisting of Ile, Lys, Arg, His, Ser, and Thr, (Xaa). m Let m be any m amino acids linked together, where m is an integer from 0 to 7, forming (Xaa). m The amino acid can be one type or two or more types.

[0030] In the amino acid sequence (2), the fourth Xaa from the N-terminus is preferably one amino acid selected from the group consisting of Ile, Lys and Thr, and more preferably Thr. In the amino acid sequence (2), as a component (Xaa) m The amino acids are preferably selected from m amino acids in the group consisting of Ile, Lys, Arg, His, Ser, Thr, Asp, Cys, Asn, Tyr, Gln, Trp and Phe, and more preferably m amino acids selected from the group consisting of Ile, Lys, Arg, His, Ser, Thr and Asp. In the amino acid sequence (2), m is preferably an integer from 0 to 6, and more preferably an integer from 1 to 6.

[0031] Amino acid sequence (3): Val-Lys-Lys-Xaa-(Xaa) k In the amino acid sequence (3), the fourth Xaa from the N-terminus is a single amino acid selected from the group consisting of Ile, Lys, and Thr. k The linkage of k amino acids selected from the group consisting of Lys, Thr, and Asp, where k is an integer from 0 to 6, forms (Xaa). k The amino acid can be one type or two or more types.

[0032] In the amino acid sequence (3), Thr is preferred as the fourth Xaa from the N-terminus. In the amino acid sequence (3), k is preferably an integer from 1 to 6.

[0033] Amino acid sequence (4): Val-Lys-Lys-Thr-Lys-Thr-(Xaa) j (Serial number 40) In amino acid sequence (4), (Xaa) j The linkage of j amino acids selected from the group consisting of Lys, Thr, and Asp, where j is an integer from 0 to 4, forms (Xaa). jThe amino acid can be one type or two or more types.

[0034] There are no restrictions on the base sequence encoding the VKKX tag. The VKKX tag base sequence is designed according to the codon table of the biosynthetic system used for tagged peptide expression (e.g., cell-free expression system).

[0035] Examples of VKKX tags and the base sequences encoding VKKX tags are shown in the table below. However, the base sequences encoding each VKKX tag are not limited to those in the table below.

[0036] [Table 1]

[0037] As details of the translation facilitation sequence, the details set forth in International Publication No. 2023 / 048290 are applicable.

[0038] There are no restrictions on the stereostructure, amino acid sequence, number of amino acid residues, types of amino acids, or base sequence encoding the expressed polypeptide. Polypeptides include those with post-translational modifications of amino acids. Examples of post-translational modifications of amino acids include phosphorylation, methylation, acetylation, glycan addition, and lipid addition. In this invention, amino acids include natural amino acids, non-natural amino acids, modified amino acids, and their derivatives.

[0039] In this invention, natural amino acids refer to the amino acids that constitute ordinary proteins, and specifically include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Natural amino acids can be natural substances or artificial products.

[0040] In this invention, non-natural amino acids refer to amino acids other than the 20 amino acids mentioned above, and also include natural and artificial products. Examples of non-natural amino acids include amino acids with halogenated acetyl groups (e.g., chloroacetylated lysine, chloroacetylated diaminobutyric acid, etc.) and N-methyl amino acids (e.g., N-methylalanine, N-methylphenylalanine).

[0041] As modified amino acids, examples include amino acids that bind to labeled compounds, i.e., labeled amino acids. Labeled compounds are substances that can be detected by biochemical, chemical, immunochemical, or electromagnetic detection methods. Examples of labeled compounds include pigment compounds, fluorescent substances, chemiluminescent substances, bioluminescent substances, enzyme substrates, coenzymes, antigenic substances, substances that bind to specific proteins, and magnetic substances. If labeled amino acids are classified by function, examples include fluorescently labeled amino acids, photoresponsive amino acids, photoconverting amino acids, and fluorescent probe amino acids.

[0042] The amino acids and labeled compounds in labeled amino acids can bind directly or via spacers. Examples of spacers include polyolefins such as polyethylene and polypropylene; polyethers such as polyethylene oxide, polyethylene glycol, and polyvinyl alcohol; and polystyrene, polyvinyl chloride, polyester, polyamide, polyimide, polyurethane, and polycarbonate.

[0043] Examples of natural amino acids, non-natural amino acids, or derivatives of modified amino acids include hydroxy acids, mercapto acids, and carboxylic acids.

[0044] In one embodiment, the polypeptide can be a polypeptide containing non-natural amino acid residues. Examples of polypeptides containing non-natural amino acid residues include polypeptides containing the non-natural amino acids exemplified above. According to the method of the present invention, even with polypeptides containing non-natural amino acids, physiological activity can be evaluated simply and at high polypeptide concentrations.

[0045] The polypeptide can be a linear polypeptide or a polypeptide containing a cyclic structure (hereinafter also referred to as a "cyclic polypeptide"). Compared with linear polypeptides, polypeptides containing a cyclic structure generally exhibit higher physiological activity. Therefore, among the polypeptides expressed, polypeptides in which the evaluation target polypeptide is partially cyclic are preferred.

[0046] The preferred cyclic polypeptide is one in which a first amino acid residue and a second amino acid residue, consisting of four or more amino acid residues, are covalently linked via the main chain or side chain. The number of amino acid residues sandwiched between the first and second amino acid residues can be, for example, 4 to 20. The covalent link can be formed through a reaction between a first functional group of the first amino acid residue and a second functional group of the second amino acid residue. The first and second functional groups can be of the same type or different types, as long as they are covalently bonded. Examples of combinations of the first and second functional groups include thiol groups and chloroacetyl groups, thiol groups and thiol groups, and carboxyl groups and amino groups in the side chain. Examples of amino acids with thiol groups include cysteine. Examples of amino acids containing a chloroacetyl group include chloroacetyldiaminobutyric acid and chloroacetylated lysine. Examples of amino acids that have a carboxyl group in their side chain include aspartic acid and glutamic acid. Examples of amino acids that have an amino group in their side chain include lysine, asparagine, and glutamine.

[0047] A polypeptide may have one ring structure or multiple ring structures. From the viewpoint of exhibiting physiological activity against cells, a higher number of ring structures in the polypeptide is preferred, while from the viewpoint of yield without cell expression, a lower number is preferred. From this viewpoint, the number of ring structures in the polypeptide is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2.

[0048] In one approach, the ring structure may include the following structures.

[0049] [Chemical Formula 2]

[0050] In the formula, X 1 and X 2 Each can be an independent linking group, and n represents an integer from 1 to 10.

[0051] The above formula can represent the binding site of two amino acid residues in a ring structure. In this case, X 1 and X 2 Linked to amino acid residues respectively, and to X 1 Linked amino acid residues and X 2 The linked amino acid residues are connected by one or more other amino acid residues to form a ring structure. As X 1 Examples of divalent organic groups include single bonds and divalent organic groups. Examples of divalent organic groups include straight-chain or branched alkylene groups with 1 to 10 carbon atoms. The number of carbon atoms in the alkylene groups with 1 to 10 carbon atoms can be 1 to 5 or 1 to 3. As X 2 Examples of divalent organic groups include single bonds and divalent organic groups. Examples of divalent organic groups include straight-chain or branched alkylene groups having 1 to 10 carbon atoms. The number of carbon atoms in the alkylene groups having 1 to 10 carbon atoms can be 1 to 5. n is 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1. As an example, the structure described above is formed by reacting an amino acid residue with a thiol group with an amino acid residue with a chloroacetylamino group. In this case, n is 1. When the amino acid residue with the thiol group is a cysteine ​​residue, X... 1X is a linear alkylene group with one carbon atom, wherein the amino acid residue with the thiol group is a homocysteine ​​residue. 1 It is a straight-chain alkylene group with two carbon atoms. When the chloroacetyl group is bonded to the side chain of a lysine residue, X... 2 X is a straight-chain alkylene group with 4 carbon atoms, in which the chloroacetyl group is bonded to the backbone of a lysine residue. 2 It is a single key.

[0052] In one approach, the polypeptide can be a cyclic polypeptide containing non-natural amino acid residues. Examples of cyclic polypeptides containing non-natural amino acid residues include polypeptides containing an amino acid with a thiol group (e.g., cysteine) and an amino acid with a chloroacetyl group (e.g., chloroacetyldiaminobutyric acid, chloroacetylated lysine) in a single molecule. In this polypeptide, the thiol group reacts with the chloroacetyl group to cyclize, thereby becoming a cyclic polypeptide.

[0053] From the viewpoint of exhibiting peptide activity, the expressed peptide preferably has 6 or more amino acid residues. From the viewpoint of expression efficiency, the peptide preferably has 600 or fewer amino acid residues, more preferably 300 or fewer, and even more preferably 150 or fewer. From this viewpoint, the expressed peptide preferably has 6 to 600 amino acid residues, more preferably 6 to 300, and even more preferably 6 to 150.

[0054] -Cell-free expression methods- Cell-free expression of peptides can be performed by translating the template nucleic acid or by transcribing and translating the template nucleic acid. Cell-free expression can use cell extracts obtained by purifying cell lysates as needed, or reconstituted solutions obtained by mixing ribosomes or other factors prepared through genetic engineering. Reconstituted solutions are preferred because they allow for control of the concentrations of various factors, enabling the setting of conditions with high efficiency in the introduction of non-natural amino acids and minimizing batch-to-batch performance variations.

[0055] Examples of cell extracts include E. coli extract, wheat germ extract, rabbit erythrocyte extract, and insect cell extract. Cell extracts typically contain ribosomes for peptide synthesis, initiation factors, and various enzymes such as tRNA. When using cell extracts for expression, various amino acids, energy sources such as ATP and GTP, and other substances used in cell-free expression, such as creatine phosphate, are usually added to the extract. Furthermore, additionally prepared ribosomes, various factors, and enzymes can be added as needed.

[0056] The reconstructed solution can be constructed from separately purified ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, GTP, ATP, translation initiation factor (IF), elongation factor (EF), release factor (RF), ribosome regeneration factor, and other factors required for cell-free peptide synthesis.

[0057] The template nucleic acid can be DNA or mRNA. It can be single-stranded or double-stranded. It can be linear or circular. The template nucleic acid can be the nucleic acid into a vector (plasmid vector, granular vector, etc.) to construct the base sequence required for polypeptide synthesis. From the viewpoint that it can be prepared rapidly and at low cost using primer extension methods, linear DNA is preferred as the template nucleic acid.

[0058] Template nucleic acids contain the base sequences required for polypeptide synthesis via cell-free expression. Template nucleic acids include promoter sequences, sequences encoding ribosome binding sites, start codons, sequences encoding affinity tags, and sequences encoding the target polypeptide. For example, in cell-free expression of E. coli factors, promoters that function in E. coli (such as the T7 promoter, lac promoter, etc.) can be used to express the target polypeptide in E. coli. Furthermore, various promoters can be positioned upstream of the sequence encoding the ribosome binding site. The ribosome binding site is located upstream of the start codon and contains the ribosome-binding sequence (SD sequence). The SD sequence is rich in adenine and guanine, for example, consisting of a sequence of the AGGAGG pattern.

[0059] In one embodiment, the template nucleic acid preferably comprises a nucleic acid sequence encoding a translation-promoting sequence immediately following the start codon. Details of the translation-promoting sequence and the base sequence encoding it are as described above.

[0060] When expressing peptides in a cell-free environment, from the perspective of improving the peptide concentration used in physiological activity evaluation, the types of codons used in the template DNA sequence are preferably optimized according to the host of cell-free expression.

[0061] In the case of cell-free expression of peptides containing non-natural amino acids, there are no restrictions on the tRNA used to introduce the non-natural amino acids, but tRNAs modified to improve introduction efficiency are preferred. Since aminoacyl-tRNAs used for introducing non-natural amino acids are easily inactivated by hydrolysis, from the viewpoint of increasing the peptide concentration used in physiological activity evaluation, a high concentration of aminoacyl-tRNA is preferred, and multiple repeated additions are preferable. Furthermore, from the viewpoint of increasing the peptide concentration used in physiological activity evaluation, it is preferable to add an aminoacylase or an artificial enzyme with the same function to the solution used in cell-free expression to regenerate the hydrolyzed aminoacyl-tRNA.

[0062] All known cell-free expression systems can be used for cell-free expression. Examples of commercially available cell-free expression systems include PUREfrex (GeneFrontier Corporation), PURExpress In VitroProtein Synthesis Kit (New England BioLabs), S30 T7 High-Yield Protein Expression System (Promega), Human Cell-Free Protein Expression System (Takara Bio Inc.), Rapid Translation System (Roche), and Expressway Cell-Free Expression System (Invitrogen).

[0063] [Process (A): Attachment Process] In step (A), the crude peptide solution after cell-free expression is contacted with a carrier to allow the pH-responsive affinity-tagged peptide to attach to the carrier. The crude peptide solution after cell-free expression is a peptide solution that has not undergone any separation or purification after cell-free expression, or even if some separation and / or purification has been performed, it contains components other than the expressed peptide.

[0064] There are no particular limitations as long as the carrier is a carrier that specifically allows the affinity label to attach. Examples include magnetic beads, agarose beads, and microbeads or plates made of resin (e.g., polystyrene) that have functional groups that specifically allow the affinity label to attach or have molecules that specifically allow the affinity label to attach fixed. The form of the carrier is not particularly limited; it can be microbeads or columns. Magnetic beads are preferred from the viewpoint of micro-solution processing because they allow for low-cost evaluation of treatable trace solutions. Examples of molecules that enable specific attachment of affinity tags include low-molecular-weight organic compounds, metal compounds (e.g., Ni-NTA), hydrophobic molecules, proteins (e.g., Strep-Tactin (registered trademark)), and antibodies (e.g., anti-PA tag antibodies, anti-FLAG tag antibodies, anti-HA tag antibodies, and anti-c-Myc antibodies).

[0065] [Process (B): Washing and Extraction Process] In step (B), the polypeptide attached to the carrier is eluted with an acidic or alkaline aqueous solution of less than 50 mM to obtain an eluent. The concentration of the acidic or alkaline aqueous solution used for elution is less than 50 mM, and more preferably less than 30 mM, and particularly preferably less than 20 mM, from the viewpoint of reducing cytotoxicity caused by the neutralized salt. From the viewpoint of elution efficiency and improving the polypeptide concentration used in physiological activity evaluation, the concentration of the acidic or alkaline aqueous solution used for elution is preferably 0.1 mM or more, more preferably 1 mM or more, and particularly preferably 10 mM. From this viewpoint, the concentration of the acidic or alkaline aqueous solution used for elution is preferably 0.1 mM or more and less than 50 mM, more preferably 1 mM or more and less than 30 mM, and even more preferably 10 mM or more and less than 20 mM.

[0066] There are no particular limitations on the type of acidic aqueous solution, but it is preferred to be an acidic aqueous solution that, after neutralization, forms a salt that does not exhibit cytotoxicity. More preferably, it is an acidic solution that, after neutralization, forms a salt typically found in culture media. Examples of salts typically found in culture media include sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, and potassium chloride. Particularly preferred is an acidic solution that, after neutralization, forms sodium chloride, which is the salt present in the largest quantity in a typical culture medium. Examples of acidic aqueous solutions that form sodium chloride after neutralization include hydrochloric acid aqueous solution. Other examples of acidic aqueous solutions include acetic acid aqueous solution, carbonic acid aqueous solution, citric acid aqueous solution, glycine aqueous solution, nitric acid aqueous solution, and sulfuric acid aqueous solution.

[0067] There are no particular limitations on the type of alkaline aqueous solution, but it is preferred to be an alkaline aqueous solution that, after neutralization, forms a salt that does not exhibit cytotoxicity. More preferably, it is an alkaline aqueous solution that, after neutralization, forms an alkaline aqueous solution containing salts typically found in culture media. Examples of the types of salts typically found in culture media are the same as those described above. Among these, an alkaline aqueous solution that, after neutralization, forms sodium chloride, which is the salt present in the largest quantity in a typical culture medium, is particularly preferred. Examples of alkaline aqueous solutions that, after neutralization, form sodium chloride include sodium hydroxide aqueous solution, sodium bicarbonate aqueous solution, and sodium carbonate aqueous solution. Examples of other alkaline aqueous solutions include calcium hydroxide aqueous solution and ammonia aqueous solution.

[0068] Other dopants (buffer components, etc.) may or may not be included in the acidic or alkaline aqueous solution used for elution. From the viewpoint of minimizing the impact on physiological activity evaluation, it is preferable to exclude other dopants.

[0069] In step (B), either an acidic or alkaline aqueous solution can be used in the elution process. From the viewpoint of inhibiting the decomposition of the target polypeptide, an acidic aqueous solution is preferred.

[0070] From the viewpoint of the removal efficiency of cytotoxic components from the crude peptide solution after cell-free expression, it is preferable to wash the carrier after step (A) and before step (B). From the viewpoint of removal efficiency of cytotoxic components and peptide yield, the number of washes is preferably 2 to 7 times, more preferably 3 to 5 times. From the viewpoint of removing cytotoxic components, it is preferable to add a surfactant to the washing solution. On the other hand, when a surfactant is added to the washing solution, it is preferable to wash with pure water just before elution to reduce the cytotoxicity of the surfactant. The type of surfactant is not limited, but from the viewpoint of reducing cytotoxicity, a nonionic surfactant is preferred, more preferably Tween20, Pluronic (registered trademark) F-68, Pluronic (registered trademark) F-127 or NP-40, and even more preferably Tween20. The concentration of the surfactant is not limited, but from the viewpoint of cytotoxicity and the removal efficiency of cytotoxic components, it is preferably 0.1% by mass to 0.0001% by mass, more preferably 0.01% by mass to 0.001% by mass.

[0071] [Process (C): Neutralization Process] In step (C), an alkaline or acidic aqueous solution is added to the eluent to neutralize any acid or base contained in the eluent obtained in step (B), and neutralization is carried out in such a way that the salt concentration formed in the neutralization reaction is less than 50 mM to obtain a polypeptide solution. If an acidic aqueous solution is used for elution in step (B), an alkaline aqueous solution is used for neutralization; if an alkaline aqueous solution is used for elution in step (B), an acidic aqueous solution is used for neutralization. In this invention, "neutralization" refers to the formation of salt through the reaction of an acid and a base, typically meaning neutralization by equal equivalent amounts of acid and base. However, the mixing of excessive amounts of acid or base is not excluded, provided that the cytotoxicity does not affect the evaluation of physiological activity (e.g., within the range that does not cause death of 80% or more, preferably 50% or more, more preferably 10% or more of the number of viable cells). From the viewpoint of pH control of the neutralized solution, the aqueous solution used for neutralization is preferably a weak base rather than a strong base, and preferably a weak acid rather than a strong acid. This is because the pH variation is smaller when the mixing ratio deviates from the desired ratio. More preferably, as a weakly alkaline aqueous solution, it is a sodium bicarbonate aqueous solution, a sodium carbonate aqueous solution, or a mixed aqueous solution of sodium bicarbonate and sodium carbonate, which, after neutralization, forms sodium chloride, the salt with the highest content in a typical culture medium. More preferably, it is a carbonated aqueous solution, which is a weakly acidic aqueous solution.

[0072] There are no particular restrictions on the concentration of the acidic or alkaline aqueous solution used for neutralization, as long as the concentration of the salt formed is less than 50 mM. It can be determined based on the acid or alkali concentration in the eluent in step (B), the volume of the eluent, and the volume of the aqueous solution used for neutralization. For example, the concentration of the acidic or alkaline aqueous solution used for neutralization can be 1 mM to 10 M, or 10 mM to 1 M.

[0073] The salt concentration of the neutralized peptide solution is less than 50 mM, preferably less than 30 mM, and more preferably less than 20 mM. Lower salt concentrations are preferred; the concentration can be 1 mM or higher, 2 mM or higher, or 5 mM or higher. From this perspective, the salt concentration of the neutralized peptide solution can be 1 mM or higher and less than 50 mM, 2 mM or higher and less than 30 mM, or 5 mM or higher and less than 20 mM.

[0074] From the perspective of reducing cytotoxicity, the pH of the neutralized peptide solution is preferably 6 to 9, and more preferably 7 to 8.

[0075] [Process (D): Evaluation Process] In step (D), the polypeptide solution obtained in step (C) is brought into contact with cells to evaluate the physiological activity of the polypeptide. There are no restrictions on the types of cells used in the evaluation of physiological activity. However, from the perspective of sensitivity, specificity, and accuracy of the evaluation, it is preferable to use cell lines modified specifically for the evaluation. Examples of cell lines modified for evaluation include cells that are forcibly expressed for specific receptors and cells with introduced reporter genes.

[0076] As a strategy to improve the evaluation concentration of peptides, a method of preparing the culture medium by mixing a large volume of purified peptide solution with a small volume of concentrated culture medium can be considered. Commercially available concentrated culture media typically have their components concentrated in equal volumes; therefore, when preparing the culture medium by diluting the purified peptide solution and the concentrated culture medium, the salt concentration in the purified peptide solution should preferably be as low as possible. This is because if the purified peptide solution contains salt, the prepared culture medium will contain a higher concentration of salt than appropriate, potentially leading to cytotoxicity.

[0077] There are no restrictions on the type of culture medium used in physiological activity evaluation; however, it is preferable to select an appropriate culture medium based on the type of physiological activity being evaluated or the type of cells used. From the viewpoint of increasing the concentration of peptides used in physiological activity evaluation, it is more preferable to use a concentrated culture medium diluted with a neutralized peptide solution.

[0078] [Other processes] In addition to cell-free expression, steps (A) to (D), and the washing step before step (B), the method of the present invention may also include additional steps. Examples of additional steps include adjusting the DNA used in cell-free expression, and quantifying the peptide concentration after cell-free expression or after step (C). From the viewpoint of obtaining high peptide yields, the method of the present invention preferably does not include other purification steps (such as size exclusion column purification) after step (C) and before physiological activity evaluation. Example

[0079] Next, specific descriptions of the embodiments of the present invention will be provided through examples, but the embodiments of the present invention are not limited to these examples.

[0080] <Example 1: Cytotoxicity Evaluation> In this embodiment, when evaluating the physiological activity of peptides synthesized via cell-free expression, the cytotoxicity of the crude peptide solution after cell-free expression, the affinity-purified eluent, and the solution obtained by neutralizing the affinity-purified eluent were evaluated. In this embodiment, to evaluate the cytotoxicity of the reaction system used in cell-free expression, a cell-free expression reaction was performed without adding template DNA, thereby preparing a cell-free expression solution that does not contain peptides. This cell-free expression solution was then subjected to affinity purification.

[0081] Chloroacetylated lysine was chosen as a non-natural amino acid. To introduce chloroacetylated lysine into the polypeptide, tRNA with the anticodon CUA and paired with the UAG codon of the mRNA was prepared by transcribing DNA of sequence number 1. The tRNA was aminoacylated using an N-chloroacetylated lysine pdCpA (5'-phospho-2'-deoxyribocytidylylriboadenosine) ester. The prepared aminoacylated tRNA was named aminoacylated tRNA (1).

[0082] Serial number 1: GTTGTAAAACGACGGCCAGTGCCAAGCTTGGGCTAATACGACTCACTATAGGGAGAGTAGTTCAATGGTAGAACGTCGGTCTCTAAAACCGAGCGTTGAGGGTTCGATTCCTTTCTCTCCCAC

[0083] As cell-free expression solutions, three reagents were used: PUREfrex2.0 (GeneFrontier Corporation, PF201-0.25-5), PURExpress In Vitro Protein Synthesis Kit (New England BioLabs, E6800S), and S30T7 High-Yield Protein Expression System (Promega, L1110). The reaction solutions were prepared with the following compositions and reacted at 37°C for 1 hour to prepare cell-free expression solutions that do not contain peptides.

[0084] • PUREfrex2.0: Mix 5.0 μL of Solution I, 0.5 μL of Solution II, 1.0 μL of Solution III, the dry body of aminoacyl-tRNA (1) (final concentration 0.5 μg / μL) and 3.5 μL of water. • PURExpress In Vitro Protein Synthesis Kit: Mix 4.0 μL of Solution A, 3.0 μL of Solution B, the dried body of aminoacyl-tRNA (1) (final concentration 0.5 μg / μL) and 3.0 μL of water. • S30 T7 High-Yield Protein Expression System: Mix 4.0 μL of S30 Premix Plus, 3.6 μL of T7 S30 Extract, dried aminoacyl-tRNA (1) (final concentration 0.5 μg / μL) and 2.4 μL of water.

[0085] The cell-free expression solution was subjected to affinity purification and neutralization treatment according to the conditions described in Table 2. In addition, regarding the conditions without affinity purification, in order to unify the dilution of the cell-free expression solution with the conditions where affinity purification was performed, 6.5 μL of the cell-free expression solution was aliquoted and mixed with 17.5 μL of water.

[0086] The following procedures were performed to determine the conditions for affinity purification. The following magnetic beads were used as the affinity carriers in affinity purification. • PA tag: MagCapture HP anti-PA tag antibody magnetic beads (FUJIFILM Wako Pure Chemical Corporation, 137-18751) • FLAG (registered trademark) label: Anti-DYKDDDDK tagged antibody magnetic beads (FUJIFILM Wako PureChemical Corporation, 011-25154) • HA Label: Pierce Anti-HA Magnetic Beads (Thermo Fisher Scientific, 88836) • His tag: HisPur Ni-NTA Magnetic Beads (ThermoFisher Scientific, 88831) • c-Myc tag: Pierce Anti-c-Myc Magnetic Beads (Thermo Fisher Scientific, 88842) • Strep label (registered trademark): MagStrep “type3” Strep-Tactin (registered trademark) beads (IBA Lifesciences, 2-1613-002)

[0087] First, the required amount of beads used in affinity purification was aliquoted and washed three times with PBS-T. Next, 8.0 μL of cell-free expression solution, 10 μL of washed microbeads, and 2.0 μL of PBS-T were mixed and shaken at room temperature (approximately 25°C) for 2 hours to perform the attachment process. Then, the mixture was placed on a magnetic rack, the supernatant was removed, and the mixture was washed three times with 100 μL of washing buffer. The types of washing buffers used are listed in Table 2. Next, the washed microbeads were placed on a magnetic rack and the supernatant was removed. Then, 25 μL of solution A was added and the mixture was shaken and mixed at room temperature for 15 minutes to perform the washing and extraction process. The eluent was placed on a magnetic rack, and 21.6 μL of the eluent supernatant was recovered and mixed with 2.4 μL of solution B to perform a neutralization process. The types of solutions A and B used are listed in Table 2. Specifically, the solutions used are as follows.

[0088] • Hydrochloric acid: Prepare 0.1 mol / L hydrochloric acid (FUJIFILM Wako Pure Chemical Corporation, 081-01111) with water to the concentration listed in Table 2 for use. • PA peptide: Prepare the PA-tagged peptide (FUJIFILM Wako Pure Chemical Corporation, 161-28681) with water to the concentrations listed in Table 2 for use. • NaOH: Prepare 0.1 mol / L sodium hydroxide (FUJIFILM Wako Pure Chemical Corporation, 194-02191) with water to the concentrations listed in Table 2 for use. • Sodium bicarbonate: Prepare sodium bicarbonate (Kanto Chemical Co., Inc., 37116-23) with water to the concentrations listed in Table 2 for use. As a comparative example of removing impurities after the elution process, regarding the conditions for size exclusion column purification of the eluent, Pierce... TM Polyacrylamide Desalting Columns (Pierce TM A polyacrylamide desalting column (Thermo Fisher Scientific, 43426) was packed into a 75 μL centrifuge column and size exclusion column purification was performed according to the manufacturer's recommended protocol.

[0089] The cytotoxicity of various solutions was evaluated using BaF3 cells stably expressing FGFR (fibroblast growth factor receptor). These cells exhibit the property of proliferating in the presence of various FGFs (fibroblast growth factor) and their similar active molecules, but dying in their absence. Stable FGFR-expressing BaF3 cells were established by introducing a vector (Gene Copoeia, EX-Y2820-M67) containing the FGFR gene into BaF3 cells using Nucleofector (Lonza), followed by drug screening using hygromycin to select cells with the introduced FGFR gene.

[0090] To allow the cell-free expression solution to act on cells at a lower dilution, the following solutions were used: 750 μL of RPMI-1640 (10×) (Sigma-Aldrich, R1145), 750 μL of Fetal Bovine Serum (Thermo Fisher Scientific, 10270-106), 75 μL of Penicillin-Streptomycin (Thermo Fisher Scientific, 15140-122), 75 μL of GlutaMAX (Thermo Fisher Scientific, 35050-061), 200 μL of Sodium Bicarbonate 7.5% solution (Thermo Fisher Scientific, 25080094), 75 μL of Folic Acid (FUJIFILM Wako Pure Chemical Corporation, 060-01802, dissolved in 1% DMSO aqueous solution to 100 μg / mL), and Heparin. To prepare RPMI medium (hereinafter referred to as "concentrated medium"), 10 μL of heparin solution (2 mg / mL) (STEM CELL, 07980), 40 μL of bFGF (a type of FGF, R&D Systems, 3718-GMP, dissolved in PBS to 0.25 μM) and 525 μL of water are mixed.

[0091] BaF3 cells stably expressing FGFR were washed twice with PBS and then cultured in a medium containing RPMI-1640 (containing L-glutamine and phenol red) (FUJIFILM Wako Pure Chemical Corporation, 189-02025) and Fetal Bovine Serum (Thermo Fisher Scientific, 10270-106) at a ratio of 10:1 to prepare 4 × 10⁶ cells. 5 cells / mL.

[0092] The solutions obtained by mixing 24 μL of each solution (crude peptide solution without cell expression, eluent obtained through affinity purification, or solution obtained by neutralizing eluent obtained through affinity purification) with 12 μL of concentrated culture medium were aliquoted into 384-well plates (Perkin Elmer, 6057300) at 15 μL each. Then, 5 μL of the above cell suspension was added to each well, and the mixture was shaken and incubated at 37°C (5% CO2) for 2 nights. As a positive control without cytotoxicity, solutions obtained by mixing 24 μL of water instead of each solution with 12 μL of concentrated culture medium were prepared in each well.

[0093] After two nights of incubation, Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water (ThermoFisher Scientific, H3570), -Cellstain-Calcein-AM solution (1 mg / mL DMSO solution) (DOJINDO, C396), and -Cellstain-PI solution (DOJINDO, P378) were added to RPMI (10% FBS) medium at 1 / 1000 volume of their final concentrations. The mixtures were then incubated at room temperature for 1 hour for live-dead staining, and the staining was performed using a confocal microscope (Yokogawa Electric Corporation, CQ1). The number of viable cells was calculated based on the imaging data, and the cell count in each well was normalized to 100, with the number of viable cells present in the wells of the positive control as a baseline. The results are shown in Table 2.

[0094] The evaluation criteria for cytotoxicity are as follows. Based on the number of cells in each well standardized to 100 viable cells in the wells of the positive control, the following values ​​are used to determine the evaluation: A through D. Evaluation A, B, or C is preferred. A…90 and above B…50 or higher and less than 90 C…20 or higher and less than 50 D…less than 20

[0095]

[0096] Under conditions without affinity purification (No. 1, 27, and 29), the cells died completely due to the toxicity of the cell expression solution, and physiological activity could not be evaluated under these conditions. Even under affinity purification conditions, the number of viable cells was significantly reduced due to the cytotoxicity of the mixed solution of solutions A and B when the concentration of the solute in solution A or solution B was high (No. 6–9). Furthermore, under conditions where neutralization was not achieved using solution B (No. 10), the number of viable cells decreased significantly due to the pH of solution A. Thus, evaluating physiological activity under near-death conditions is extremely difficult. A decrease in the number of viable cells was observed under the condition that solution A was a PA peptide solution (No. 12). While physiological activity evaluation is not impossible under these conditions, as described later in Example 2, this method of competitively eluting the target analyte using peptides has low elution efficiency, resulting in a significantly reduced evaluation concentration, making it difficult to use for physiological activity evaluation. Increasing the concentration of PA peptides to improve elution efficiency would increase the toxicity caused by the high concentration of PA peptides, making it difficult to balance high elution efficiency with low toxicity. Therefore, physiological activity evaluation using competitive elution is extremely difficult. Under the conditions of size exclusion column purification (No. 26) to remove dopant after elution, the number of viable cells was maintained, but as described later in Example 2, there was significant peptide loss during the dopant removal process, making it difficult to achieve both high evaluation concentration and low toxicity. The number of viable cells was maintained under the conditions of low concentrations of acid or base in solutions A and B (No. 2–5, 11, 13–25, 28 and 30), indicating that physiological activity evaluation could be performed under these conditions. Comparing wash No. 2 (washed with water before extraction) with wash No. 11 (washed with PBS-T), wash No. 2 showed a higher number of viable cells. This indicates that washing with water before extraction can remove trace amounts of PBS-T toxicity, thereby further inhibiting the reduction of viable cell count. Based on these results, washing with water before extraction can further improve the accuracy and sensitivity of physiological activity evaluation.

[0097] <Example 2: Evaluation of the physiological activity of affinity-purified peptides> In this embodiment, the peptide of sequence number 2, which has bFGF-like physiological activity, underwent affinity purification and neutralization after cell-free expression. The yield during affinity purification and neutralization, as well as the physiological activity of the peptide solution obtained after affinity purification and neutralization, were evaluated. The yield during affinity purification and neutralization is an important performance indicator for evaluating physiological activity at high peptide concentrations.

[0098] Serial Number 2: HC-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-XV (X represents a chloroacetylated lysine residue that is a non-natural amino acid residue. This polypeptide is a two-ring polypeptide in which a thioether bond is formed between the side chains of the first cysteine ​​residue and the first chloroacetylated lysine residue starting from the N-terminus, and also between the side chains of the second cysteine ​​residue and the second chloroacetylated lysine residue starting from the N-terminus.)

[0099] Cell-free expression of the peptide was performed according to the following steps. First, fusion peptides (Sequence Nos. 11-16) were designed by sequentially linking a translation-promoting sequence (Sequence No. 3), a peptide of Sequence No. 2, a HiBiT tag for concentration quantification (Sequence No. 4), and an affinity tag for affinity purification (any one of Sequence Nos. 5-10).

[0100] Serial Number 3: VKKTKT Serial Number 4: VSGWRLFKKIS Serial Number 5 (PA Tag): GVAMPGAEDDVV Serial Number 6 (FLAG Tag): DYKDDDDK Serial Number 7 (HA Tag): YPYDVPDYA Serial number 8 (His tag): HHHHHH Serial number 9 (c-Myc tag): EQKLISEEDL Serial Number 10 (Strep Tag): WSHPQFEK Serial number 11 (PA-tagged fusion peptide): MVKKTKTH-C-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-X-VGSGVSGWRLFKKISSGSGVAMPGAEDDVV Serial number 12 (flag-tagged fusion peptide): MVKKTKTH-C-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-X-VGSGVSGWRLFKKISSGSDYKDDDDK Serial number 13 (HA-tagged fusion peptide): MVKKTKTH-C-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-X-VGSGVSGWRLFKKISSGSYPYDVPDYA Serial number 14 (His-tagged fusion peptide): MVKKTKTH-C-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-X-VGSGVSGWRLFKKISSGSHHHHHH Serial number 15 (fusion peptide with c-Myc tag): MVKKTKTH-C-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-X-VGSGVSGWRLFKKISSGSEQKLISEEDL Serial number 16 (strep-tagged fusion peptide): MVKKTKTH-C-SYERLQFHGHEAPFRV-X-VSH-C-SYERLQFHGHEAPFRV-X-VGSGVSGWRLFKKISSGSWSHPQFEK

[0101] Next, template DNA sequences (sequence numbers 11-16) encoding the aforementioned fusion peptides were designed with the T7 promoter sequence, Shine-Dalgarno sequence, and start codon (ATG) at the 5' end, and a set of stop codons at the 3' end (sequence numbers 17-22). In the case where the template DNA sequence contains a triplet TAG codon, this codon was mapped to the codon for chloroacetylated lysine. Therefore, the thiol group of cysteine ​​in the peptide encoded by the aforementioned DNA sequence spontaneously forms a thioether bond with the chloroacetyl group of chloroacetylated lysine, resulting in a polypeptide with two rings.

[0102] Sequence number 17 (template DNA for fusion peptide with PA tag): gaaattaatacgactcactataggggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGTTAAAAAAACAAAAACACATTGCCTCTACGAACGTCTGC AGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTTTCGCACTGTAGCTATGAGCGCTTGCAATTTCACGGCCACGAGGCACCCTTTCGCGTATAGGTAGGAAGTGGTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCAGCGGCAGT GGAGTTGCAATGCCTGGTGCAGAAGATGATGTAGTA taatga

[0103] Serial number 18 (template DNA for the fusion peptide with FLAG tag): gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGTTAAAAAAACAAAAACACATTGCTCTTACGAACGTCTGCAGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTTTCGCACTGTAGCTATGAGCGCTTGCAATTTCACGGCCACGAGGCACCCTTTCGCGTATAGGTAGGAAGTGGTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCAGCGGCAGT GACTACAAAGACGACGACGACAAA taatga

[0104] Serial number 19 (template DNA for the fusion peptide with HA tag): gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGTTAAAAAAACAAAAACACATTGCTCTTACGAACGTCTGCAGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTTTCGCACTGTAGCTATGAGCGCTTGCAATTTCACGGCCACGAGGCACCCTTTCGCGTATAGGTAGGAAGTGGTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCAGCGGCAGT TACCCGTACGACGTTCCGGACTACGCT taatga

[0105] Serial number 20 (template DNA for the fusion peptide with His-tag): gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGTTAAAAAAACAAAAACACATTGCTCTTACGAACGTCTGCAGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTTTCGCACTGTAGCTATGAGCGCTTGCAATTTCACGGCCACGAGGCACCCTTTCGCGTATAGGTAGGAAGTGGTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCAGCGGCAGT CATCATCATCATCATCAT taatga

[0106] Serial number 21 (template DNA for the fusion peptide with c-Myc tag): gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGTTAAAAAAACAAAAACACATTGCTCTTACGAACGTCTGCAGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTTTCGCACTGTAGCTATGAGCGCTTGCAATTTCACGGCCACGAGGCACCCTTTCGCGTATAGGTAGGAAGTGGTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCAGCGGCAGT GAACAGAAACTGATCTCTGAAGAAGACCTG taatga

[0107] Sequence number 22 (template DNA for fusion peptide with Strep tag): gaaattaatacgactcactataggggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGTTAAAAAAACAAAAACACATTGCTCTTACGAACGTCTG CAGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTTTCGCACTGTAGCTATGAGCGCTTGCAATTTCACGGCCACGAGGCACCCTTTCGCGTATAGGTAGGAAGTGGTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCAGCGGCAGT TGGTCTCATCCGCAGTTCGAAAAA taatga

[0108] In the above sequences, the DNA sequence encoding the fusion peptide is recorded in uppercase letters, and the sequences assigned at the 5' and 3' ends are recorded in lowercase letters. Furthermore, the DNA sequence encoding the affinity tag for purification is underlined.

[0109] The template DNA was prepared by two-stage overlap amplification PCR. In the first stage of PCR, DNA from sequence number 23, sequence number 24, sequence number 25, and sequence number 26 were mixed at concentrations of 0.3 μmol / L, 0.3 μmol / L, 0.05 μmol / L, and 0.05 μmol / L, respectively. In the presence of PrimeSTARMax (TaKaRa, R045B), the ligation process was repeated 27 times at 98°C / 10 s, 39°C / 5 s, and 72°C / 5 s, followed by three cycles of 98°C / 10 s, 58°C / 5 s, and 72°C / 5 s. This ligation of sequence number 25 and sequence number 26 was achieved. The ligated DNA was then purified and diluted to a concentration of 50 ng / μL.

[0110] Serial number 23: GAAATTAATACGACTCACTATAGG Serial number 24: CGAAACCTAAACACGGAA Serial number 25: GAAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATGGTTAAAAAAACAAAAAC Serial number 26: CGAAACCTAAACACGGAACGGAGCTTCATGACCATGGAACTGCAGACGTTCGTAAGAGCAATGTGTTTTTGTTTTTTTAAC

[0111] In the second stage of PCR, four DNAs—DNA of sequence number 23, any one of DNAs from sequences 27–32, any one of DNAs from sequences 33–38, and the purified product from stage 1—were mixed to concentrations of 0.3 μmol / L, 0.3 μmol / L, 0.0025 μmol / L, and 0.4 ng / μL, respectively. Under PrimeSTAR Max (TaKaRa, R045B), the mixture was repeatedly subjected to 30 cycles of 98°C / 10 sec, 58°C / 5 sec, and 72°C / 5 sec, thereby ligating DNAs of sequences 33–38 to the purified product from stage 1, thus obtaining template DNA (sequence numbers 17–22). At this point, DNAs of sequences 27–32 and 33–38 were combined using the same tag types for ligation. The obtained template DNA was purified and diluted to 50 ng / μL.

[0112] Serial number 27 (PA tag): TCATTATACTACATCATCTTCTGCACCAG Serial Number 28 (FLAG Tag): TCATTATTTGTCGTCGTCG Serial number 29 (HA tag): TCATTAAGCGTAGTCCGGAAC Serial number 30 (His tag): TCATTAATGATGATGATGATGA Serial number 31 (c-Myc tag): TCATTACAGGTCTTCTTCAGAGATCAG Serial number 32 (Strep tag): TCATTATTTTTCGAACTGCG Serial number 33 (PA label): CATCATCTTCTGCACCAGGCATTGCAACTCCACTGCCGCTGCTAATCTTCTTGAATAATCGCCATCCACTTACACCACTTCCTACCTATACGCGAAAGGGTGCCTCGTGGCCGTGAAATTGCAAGCGCTCATAGCTACAGTGCGAAACCTAAACACGGAA Serial number 34 (FLAG tag): TCATTATTTGTCGTCGTCGTCTTTGTAGTCACTGCCGCTGCTAATCTTCTTGAATAATCGCCATCCACTTACACCACTTCCTACCTATACGCGAAAGGGTGCCTCGTGGCCGTGAAATTGCAAGCGCTCATAGCTACAGTGCGAAACCTAAACACGGAA Serial number 35 (HA tag): ATTAAGCGTAGTCCGGAACGTCGTACGGGTAACTGCCGCTGCTAATCTTCTTGAATAATCGCCATCCACTTACACCACTTCCTACCTATACGCGAAAGGGTGCCTCGTGGCCGTGAAATTGCAAGCGCTCATAGCTACAGTGCGAAACCTAAACACGGAA Serial number 36 (His tag): TCATTAATGATGATGATGATGATGACTGCCGCTGCTAATCTTCTTGAATAATCGCCATCCACTTACACCACTTCCTACCTATACGCGAAAGGGTGCCTCGTGGCCGTGAAATTGCAAGCGCTCATAGCTACAGTGCGAAACCTAAACACGGAA Serial number 37 (c-Myc tag): ACAGGTCTTCTTCAGAGATCAGTTTCTGTTCACTGCCGCTGCTAATCTTCTTGAATAATCGCCATCCACTTACACCACTTCCTACCTATACGCGAAAGGGTGCCTCGTGGCCGTGAAATTGCAAGCGCTCATAGCTACAGTGCGAAACCTAAACACGGAA Serial number 38 (Strep tag): TCATTATTTTTCGAACTGCGGATGAGACCAACTGCCGCTGCTAATCTTCTTGAATAATCGCCATCCACTTACACCACTTCCTACCTATACGCGAAAGGGTGCCTCGTGGCCGTGAAATTGCAAGCGCTCATAGCTACAGTGCGAAACCTAAACACGGAA

[0113] The prepared template DNA was subjected to cell-free expression, affinity purification, and neutralization according to the conditions described in Table 3.

[0114] Similar to Example 1, cell-free expression of peptides was performed using three devices: PUREfrex2.0 (GeneFrontier Corporation, PF201-0.25-5), PURExpress In Vitro Protein Synthesis Kit (New England BioLabs, E6800S), and S30T7 High-Yield Protein Expression System (Promega, L1110). Reaction solutions were prepared with the following compositions and incubated at 37°C for 1 hour to achieve cell-free peptide expression.

[0115] • PUREfrex2.0: Mix 5.0 μL of Solution I, 0.5 μL of Solution II, 1.0 μL of Solution III, the dry body of aminoacyl-tRNA (1) (final concentration 0.5 μg / μL), 2.0 μL of template DNA and 1.5 μL of water. • PURExpress In Vitro Protein Synthesis Kit: Mix 4.0 μL of Solution A, 3.0 μL of Solution B, a dry body of aminoacyl-tRNA (1) (final concentration 0.5 μg / μL), 2.0 μL of template DNA and 1.0 μL of water. • S30 T7 High-Yield Protein Expression System: Mix 4.0 μL of S30 Premix Plus, 3.6 μL of T7 S30 Extract, dried aminoacyl-tRNA (1) (final concentration 0.5 μg / μL), 2.0 μL of template DNA and 0.4 μL of water.

[0116] The cell-free expressed peptides were subjected to affinity purification and neutralization treatment using the same procedures as in Example 1. For the conditions without affinity purification, similar to Example 1, to ensure consistency with the conditions where affinity purification was performed, 6.5 μL of the cell-free expression solution was aliquoted and mixed with 17.5 μL of water.

[0117] Regarding the peptide concentrations after affinity purification and neutralization, the Nano Glo HiBiT Lytic Detection System (Promega, N3040) and a peptide of known concentration (Sequence No. 39) were used. The affinity-purified and neutralized products diluted 100-fold with Can Get Signal Immunoreaction Enhancer Solution I (TOYOBO, NKB-101) were determined according to the standard protocol of the Nano Glo HiBiT Lytic Detection System. The obtained peptide concentrations are listed in Table 3 with two significant figures. Peptide No. 39, after chemical synthesis, was dissolved in PBS(-) at 500 μM and the solution was appropriately diluted before use.

[0118] Serial Number 39: EQKLISEEDLGGSVSGWRLFKKIS

[0119] The peptide yields during affinity purification and neutralization treatments were calculated based on the peptide concentrations under conditions where affinity purification was not performed, and are recorded in Table 3. The evaluation criteria for peptide yield during affinity purification and neutralization are as follows. Peptide yields during affinity purification and neutralization are classified as A through D based on the following proportions. Evaluation A, B, or C is preferred. A…more than 50% B…10% or more but less than 50% C…5% or more but less than 10% D…less than 5%

[0120]

[0121] The yield of the peptides varied slightly depending on the label type, but the required yield for evaluation was achieved under the conditions of low concentrations of acid or base in solutions A and B (No. 2–5, 11, 13–15, 22–25, 28, and 30). Under the condition that solution A is a PA peptide solution (No. 12), the elution efficiency is low, resulting in a significant decrease in the evaluation concentration, making it difficult to use for physiological activity evaluation. Under the condition of size exclusion column purification (No. 26) to remove dopants after elution, there is a lot of peptide loss in the dopant removal process, resulting in a significant decrease in the evaluation concentration, making it difficult to use for physiological activity evaluation.

[0122] Similar to Example 1, the physiological activity of peptides subjected to affinity purification and neutralization under various conditions was evaluated using BaF3 cells stably expressing FGFR (fibroblast growth factor receptor). These cells possess the property of proliferating in the presence of various FGFs (fibroblast growth factors) and their similar active molecules, but dying in their absence; therefore, they exhibit proliferative activity in the presence of peptides with bFGF-like physiological activity. To allow the cell-free expression solution to act on cells at a lower dilution, the following solutions were used: 750 μL of RPMI-1640 (10×) (Sigma-Aldrich, R1145), 750 μL of Fetal Bovine Serum (Thermo Fisher Scientific, 10270-106), 75 μL of Penicillin-Streptomycin (Thermo Fisher Scientific, 15140-122), 75 μL of GlutaMAX (Thermo Fisher Scientific, 35050-061), 200 μL of Sodium Bicarbonate 7.5% solution (Thermo Fisher Scientific, 25080094), 75 μL of Folic Acid (FUJIFILM Wako Pure Chemical Corporation, 060-01802, dissolved in 1% DMSO aqueous solution to 100 μg / mL), and Heparin. To prepare RPMI medium (hereinafter referred to as "concentrated medium"), 10 μL of heparin solution (2 mg / mL) (STEM CELL, 07980) and 565 μL of water were mixed. This concentrated medium was three times more concentrated than the usual composition. To evaluate the bFGF-like physiological activity of the peptides, this concentrated medium did not contain bFGF, unlike the concentrated medium in Example 1.

[0123] BaF3 cells stably expressing FGFR were washed twice with PBS and then cultured in a medium containing RPMI-1640 (containing L-glutamine and phenol red) (FUJIFILM Wako Pure Chemical Corporation, 189-02025) and Fetal Bovine Serum (Thermo Fisher Scientific, 10270-106) at a ratio of 10:1 to prepare 4 × 10⁶ cells. 5 cells / mL.

[0124] The solutions obtained by mixing 24 μL of peptides that had undergone affinity purification and neutralization under various conditions with 12 μL of concentrated culture medium were aliquoted into 384-well plates (Perkin Elmer, 6057300) at 15 μL each. Then, 5 μL of the aforementioned cell suspension was added to each well, and the mixture was shaken and incubated at 37°C (5% CO2) for two nights. As a negative control lacking physiological activity, solutions were prepared by aliquoting 24 μL of water instead of 12 μL of concentrated culture medium at 15 μL each.

[0125] After two nights of culture, Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water (ThermoFisher Scientific, H3570), -Cellstain-Calcein-AM Solution (1 mg / mL DMSO Solution) (DOJINDO, C396), and -Cellstain-PI Solution (DOJINDO, P378) were added to RPMI (10% FBS) medium at 1 / 1000 volume of their final concentrations. The mixtures were incubated at room temperature for 1 hour for viability staining, and imaging was performed using a confocal microscope (Yokogawa Electric Corporation, CQ1). The number of viable cells was calculated based on the imaging data and recorded in Table 4.

[0126]

[0127] In each case, the number of live cells increased more than 10-fold compared to the negative control, indicating that the physiological activity of cell-free expressed peptides can be evaluated using the method of the present invention.

[0128] The entire contents of the invention of Japanese Patent Application No. 2023-064440, filed on April 11, 2023, are incorporated herein by reference. All documents, patent applications, and technical standards described in this specification are incorporated herein by reference to the same extent as those specifically and separately described and incorporated herein by reference.

Claims

1. A method for preparing a polypeptide solution for evaluating physiological activity, wherein, The method includes the following steps: (A) The crude solution of the cell-free expressed peptide is contacted with the carrier so that the peptide with a pH-responsive affinity tag attaches to the carrier; (B) The polypeptide attached to the carrier is eluted with an acidic or alkaline aqueous solution of less than 50 mM to obtain an eluent. and (C) To neutralize the acid or base contained in the eluent, an alkaline or acidic aqueous solution is added to the eluent, and the neutralization is carried out in such a way that the salt concentration formed in the neutralization reaction is less than 50 mM to obtain a polypeptide solution.

2. A method for evaluating the physiological activity of peptides, wherein, The method includes the following steps: Prepare a polypeptide solution using the method of claim 1; and The polypeptide solution was brought into contact with cells to evaluate the physiological activity of the polypeptide.

3. The method according to claim 1 or 2, wherein, The polypeptide contains non-natural amino acid residues.

4. The method according to claim 1 or 2, wherein, The polypeptide contains one or more ring structures.

5. The method according to claim 4, wherein, The ring structure includes the following structure. [Chemical Formula 1] In the formula, X 1 and X 2 Each can be an independent linking group, and n represents an integer from 1 to 10.

6. The method according to claim 4, wherein, The polypeptide contains 1 to 4 ring structures.

7. The method according to claim 1 or 2, wherein, In step (B), an acidic aqueous solution of less than 50 mM is used, and in step (C), an alkaline aqueous solution is used.

8. The method according to claim 7, wherein, The acidic aqueous solution is a hydrochloric acid aqueous solution.

9. The method according to claim 7, wherein, The alkaline aqueous solution is an aqueous solution of sodium bicarbonate, an aqueous solution of sodium carbonate, or a mixed aqueous solution of sodium bicarbonate and sodium carbonate.

10. The method according to claim 1 or 2, wherein, The carrier is a magnetic bead.

11. The method according to claim 1 or 2, wherein, The affinity label is a FLAG label, HA label, His label, c-Myc label, V5 label, Strep label, or PA label.

12. The method according to claim 1 or 2, wherein, The method further includes, prior to step (A), a step of expressing the polypeptide in a cell-free manner.

13. The method according to claim 1 or 2, wherein, The method, after (A) and before (B), further includes a step of washing the carrier with water.

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