Method for cyclizing and purifying a target protein from a fusion protein containing the target protein, method for producing a cyclized target protein from a fusion protein containing the target protein, and fusion protein for use in cyclizing and purifying a target protein

Abstract

To provide a purification method capable of performing removal of tag peptide in combination with purification of target protein.SOLUTION: A method for cyclizing and purifying target protein from fusion protein including target protein, where the fusion protein includes a tag domain, a target protein and inteins C, N, the intein C is arranged on an N-terminal side of the target protein, the intein N is arranged on a C-terminal side of the target protein, the tag domain is arranged on an N-terminal side of the intein C and / or a C-terminal side of the intein N, and the method includes the steps of: allowing a carrier including a binding partner of the tag domain to hold the fusion protein by forming a composite with the carrier; separating a cyclized target protein from the intein C, the intein N and the tag domain by binding between the intein C and the intein N; and recovering the separated circularization target protein.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for circularizing and purifying a target protein from a fusion protein containing the target protein, a method for producing a circularized target protein from a fusion protein containing the target protein, and a fusion protein for use in circularizing and purifying a target protein. [Background technology]

[0002] Protein purification is performed by utilizing the affinity between a tag peptide, such as a His tag, attached to a protein and a binding partner that specifically binds to the tag peptide. However, when purifying a protein using affinity, the tag peptide is purified in a state where it is attached to the target protein. When using the protein as a pharmaceutical, there is a concern that an immune response against the tag peptide may be induced, and therefore it is desirable that the final product does not contain the tag peptide. Furthermore, depending on the type of tag peptide, the introduction of molecules that bind to the tag peptide, such as heavy metal ions in the case of a His tag, may pose a problem in applying for pharmaceutical approval. For this reason, a purification method that can remove the tag peptide is desired. Summary of the Invention [Problem to be solved by the invention]

[0003] One possible method for removing the tag peptide is to use an enzyme that removes the tag peptide, but this requires a step of purifying the target protein again after removing the tag peptide, which increases the number of steps.

[0004] Furthermore, a method using Npu intein has been proposed as another method for removing tag peptides, and it has been confirmed that the His tag can be removed using a VHH antibody with a His tag attached.

[0005] However, splicing occurs in the presence of both intein C and intein N. Therefore, when attempting to remove the tag peptide from a recombinant protein, the tag peptide is removed and the protein is cyclized immediately after protein synthesis, making purification using the tag peptide difficult.

[0006] Therefore, an object of the present invention is to provide a purification method that allows removal of tag peptides to be carried out in conjunction with purification of target proteins. [Means for solving the problem]

[0007] In order to achieve the above object, the method of the present invention is a method for cyclizing and purifying a target protein from a fusion protein containing the target protein (hereinafter also referred to as a "purification method"), which comprises the steps of: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; contacting the fusion protein with a carrier comprising a binding partner capable of binding to the tag domain, and allowing the tag domain to bind to the binding partner to form a complex, thereby retaining the fusion protein on the carrier; allowing the intein C and the intein N to bind to each other under conditions that allow a binding reaction between the intein C and the intein N to occur, and separating the circularized target protein from the intein C, the intein N, and the tag domain; and recovering the separated, circularized target protein.

[0008] The method of the present invention is a method for producing a cyclized target protein from a fusion protein containing the target protein (hereinafter also referred to as a "production method"), which comprises the steps of: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; circularizing the target protein in the fusion protein; The cyclization is carried out by the purification method of the present invention.

[0009] The fusion protein of the present invention is a fusion protein for use in cyclization and purification of a target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; The intein is an intein in which a binding reaction occurs condition-dependently. [Effects of the Invention]

[0010] According to the present invention, removal of the tag domain can be performed in conjunction with purification of the target protein. [Brief explanation of the drawings]

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

[0012] <Definition> As used herein, "protein" refers to a peptide polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The polymer may be, for example, linear, branched, or cyclic. The protein may also be referred to as a peptide or polypeptide.

[0013] As used herein, the term "fusion protein" refers to a protein in which two or more different or heterologous proteins are partially or entirely bound (linked) via peptide bonds. The fusion protein may be a natural fusion protein or an artificial fusion protein. Examples of the artificial fusion protein include proteins designed by genetic engineering techniques.

[0014] As used herein, the term "tag domain" refers to a polypeptide that specifically binds to another molecule. Since the tag domain constitutes a part of the fusion protein, it can also be called a protein tag, a peptide tag, or a tag peptide.

[0015] As used herein, the term "binding partner" refers to a substance that has specific binding affinity with the tag domain.

[0016] As used herein, "target protein" refers to a protein that is the target of circularization and / or purification.

[0017] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Immunoglobulin genes include genes encoding constant regions such as κ, λ, α (including α1 and α2), γ (including γ1, γ2, γ3, and γ4), δ, ε, and μ, as well as genes capable of encoding numerous immunoglobulin variable regions such as V regions, D regions, and J regions. The antibody comprises, for example, a heavy chain and a light chain. The light chain includes κ and λ, constituting the κ chain and the λ chain, respectively. The heavy chain includes γ, μ, α, δ, or ε, constituting the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The antibody may be a typical immunoglobulin (antibody) structural unit composed of a tetramer. In this case, the antibody is composed of two identical pairs of polypeptide chains, each pair consisting of one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa), and the N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids that is primarily responsible for antigen recognition.

[0018] As used herein, "antigen-binding fragment" refers to a portion or partial polypeptide comprising the antigen-binding site of an antibody. The antigen-binding fragment can be obtained by chemical or enzymatic treatment of an antibody. The antigen-binding fragment can also be obtained by recombinant means. Examples of the antigen-binding fragment include Fab, Fab', F(ab')2, Fc, and / or Fv fragments, and derivatives thereof.

[0019] As used herein, the term "multispecific antibody" refers to an antibody or antibody derivative having specificity for more than one antigen and / or more than one epitope.

[0020] As used herein, "intein" refers to an autocatalytic enzyme that has both protein protease activity and protein ligase activity. For example, an intein is a protein that excises its own amino acid sequence from a protein by protein splicing and ligates the remaining amino acid sequence (extein) via a peptide bond. The "intein" can also be referred to as, for example, a protein intron.

[0021] As used herein, the term "split intein" refers to an intein that contains two complementary half inteins, intein C and intein N, which tightly bind to form an active intein and exhibit enzymatic activity. The split intein is also called a split intein.

[0022] As used herein, "complementary inteins" refers to a pair of inteins or split inteins, intein C and intein N.

[0023] As used herein, "intein C" refers to an intein polypeptide that shares homology with the C-terminal portion of an intein, which binds to a complementary intein N to form an active intein.

[0024] As used herein, "intein N" refers to an intein polypeptide that shares homology with the N-terminal portion of an intein, which binds to a complementary intein C to form an active intein.

[0025] As used herein, the term "salt" refers to an electrolyte that can be ionized in an aqueous solvent. The aqueous solvent refers to a solvent that contains water.

[0026] As used herein, a "solubilization domain" refers to a polypeptide that, when fused to a fusion protein, increases (e.g., increases or promotes) the expression level of the fusion protein expressed in a transformant compared to when the fusion protein is expressed as a fusion protein without the solubility domain.

[0027] As used herein, "cyclization" refers to the formation of an amide bond (peptide bond) between the N-terminal amino acid and the C-terminal amino acid of a protein, causing the protein to become circular, and / or the state in which the protein has formed a cyclic structure.

[0028] As used herein, "purified" means identifying and separating, recovering from components in their natural state, being identified and separated, and / or recovered from components in their natural state. The "purification" can be performed, for example, by obtaining at least one purification step. The purification can also be referred to as isolation.

[0029] As used herein, "separation" refers to the separation of a target substance from a substance containing the target substance and / or the state of being separated. Furthermore, when "separation" is used in conjunction with a target protein, "separation" refers to the excision of the target protein from a fusion protein and / or the state of the target protein being excised from the fusion protein. Separation can also be referred to as liberation.

[0030] As used herein, "support" refers to a substance capable of carrying (eg, holding, positioning, immobilizing, or binding) a binding partner.

[0031] As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "nucleic acid" is used in combination with a specific protein, the "nucleic acid" refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA. The nucleic acid may be, for example, single-stranded or double-stranded. The nucleic acid can be interchangeably referred to as a "polynucleotide" or a "nucleic acid molecule."

[0032] As used herein, the term "host" refers to a cell and / or an individual into which exogenous nucleic acid is introduced. When the host is a cell, the host can also be referred to as a host cell.

[0033] As used herein, the terms "vector" and "expression vector" are used interchangeably. in vitro or in vivo In the present specification, the term "vector" refers to a recombinant plasmid or virus containing a nucleic acid to be delivered to a host or host cell. The term "vector" and "expression vector" includes viral vectors and non-viral vectors.

[0034] As used herein, the term "transformant" refers to a host into which exogenous nucleic acid has been introduced.

[0035] The present invention will be described below using examples, but the present invention is not limited to the following examples and can be practiced with any modifications. Furthermore, each description in the present invention can be mutually incorporated unless otherwise specified. In this specification, when the expression "to" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0036] <Method for cyclization and purification of target proteins> In one aspect, the present invention provides a purification method that allows removal of a tag peptide and purification of a target protein to be performed simultaneously. The purification method of the present invention is a method for circularizing and purifying a target protein from a fusion protein containing the target protein, wherein the fusion protein comprises a tag domain, the target protein, and an intein, the intein comprising intein C and intein N, wherein in the fusion protein, the intein C is located at the N-terminus of the target protein, the intein N is located at the C-terminus of the target protein, and the tag domain is located at the N-terminus of the intein C and / or the C-terminus of the intein N, the method comprising the steps of: contacting the fusion protein with a carrier comprising a binding partner capable of binding to the tag domain, and allowing the tag domain to bind to the binding partner to form a complex, thereby retaining the fusion protein on the carrier; binding the intein C to the intein N under conditions in which a binding reaction between the intein C and the intein N occurs, and separating the circularized target protein from the intein C, the intein N, and the tag domain; and recovering the separated circularized target protein. In the fusion protein used in the purification method of the present invention, a tag domain is located at the N-terminus of the C intein and / or the C-terminus of the N intein. Therefore, in the purification method of the present invention, the tag domain-binding partner bond is utilized. When the intein is allowed to exert its enzymatic activity while the fusion protein is retained on the carrier, the region other than the intein and tag domain is excised and cyclized by the intein. In other words, the target protein is cyclized and excised from the fusion protein as an extein. Meanwhile, the intein and tag domain remain retained on the carrier due to the tag domain-binding partner bond. Therefore, according to the present invention, removal of a tag domain such as a tag peptide and purification of a target protein can be performed simultaneously. The purification method of the present invention can be applied to the following descriptions of the fusion protein, nucleic acid, expression vector, transformant, method for producing a transformant, and method for producing a fusion protein of the present invention.

[0037] The present inventors conceived the idea that the target protein could be cyclized and purified by controlling the enzymatic activity of the intein and allowing the intein's protease and ligase activities to be exerted on a fusion protein containing the intein, the tag domain, and the target protein while the fusion protein is held on a support via the tag domain and its binding partner. After extensive research, the present inventors discovered that the target protein could be cyclized and purified by arranging the intein, the tag domain, and the target protein in the aforementioned order in the fusion protein and binding the intein C and intein N, thereby establishing the present invention. Specifically, the purification method of the present invention is presumed to cyclize and purify the target protein by the mechanism described below, as shown in Figure 1 . However, the present invention is not limited to the mechanism described below. In the purification method of the present invention, the fusion protein contains the intein C, the target protein, the intein N, and the tag domain, as shown in Figure 1(A). In the following description, the intein C, the target protein, the intein N, and the tag domain are arranged in this order from the N-terminus of the fusion protein. However, the same applies when the tag domain is added to the N-terminus of the intein C. First, in the purification method of the present invention, the fusion protein shown in FIG. 1(A) is contacted with a carrier containing a binding partner to which the tag domain specifically binds. As shown in FIG. 1(A), the fusion protein may be in a state in which the intein C and intein N are unbound, or in an inactive state in which the intein C and intein N are bound, or a mixture of both. As a result, the fusion protein forms a complex on the carrier through binding between the tag domain and the binding partner, as shown in FIG. 1(B), and is retained on the carrier (FIG. 1(C)). In this state, when the complex is placed under conditions that allow a binding reaction between the intein C and the intein N to occur, the tightly bound intein C and intein N (intein complex) exhibits enzymatic activity.As a result, as shown in Figure 1(D), peptide bond recombination occurs between the intein C and intein N and the target protein, linking the N-terminal amino acid and the C-terminal amino acid of the target protein by a peptide bond, and the target protein is cyclized (Figure 1(E)). Meanwhile, the intein C, intein N, and tag peptide are retained on the carrier by the binding between the tag domain and the binding partner. Therefore, in the purification method of the present invention, the target protein can be purified by recovering the cyclized target protein. Furthermore, if the arrangement of the intein C and intein N in the fusion protein is swapped, the target protein will be retained on the carrier via the tag domain after the binding reaction. Therefore, in the purification method of the present invention, it is presumed that the target protein can be cyclized and purified by configuring each domain in the fusion protein as described above.

[0038] In the purification method of the present invention, the fusion protein can be prepared, for example, by genetic engineering techniques, as described below in the method for producing a fusion protein of the present invention. Therefore, the purification method of the present invention may include, for example, a preparation step of preparing the fusion protein prior to the holding step. In this case, the purification method of the present invention, for example, subjects the sample containing the fusion protein obtained in the preparation step to the holding step. The preparation method in the preparation step can be based on the explanations of the fusion protein, nucleic acid, transformant, and method for producing a fusion protein of the present invention described below.

[0039] In the retention step, the fusion protein is brought into contact with a carrier containing a binding partner capable of binding to the tag domain, and the tag domain and the binding partner are allowed to bind to form a complex, thereby retaining the fusion protein on the carrier (FIGS. 1(B) to 1(C)). The tag domain can specifically bind to the binding partner contained in the carrier. Therefore, in the retention step, a complex between the fusion protein and the binding partner is formed via specific binding between the tag domain in the fusion protein and the binding partner on the carrier, and this complex can be retained on the carrier. The retention can also be referred to as, for example, binding.

[0040] In the holding step, the intein C and the intein N may or may not form an intein complex with a complementary intein. If the intein C and the intein N form an intein complex in the holding step, the enzymatic activity of the complex is preferably inactive.

[0041] In the retention step, the fusion protein contacted with the carrier may be a purified or crudely purified fusion protein, a transformant expressing the fusion protein, or a processed product of the transformant. Examples of the processed product include a lysate, extract, disrupted product, protein preparation, or crude or purified product thereof. The processed product may be, for example, a product that has been subjected to at least one purification treatment.

[0042] In the fusion protein, the tag domain can be appropriately selected depending on the type of the binding partner. The tag domain can be, for example, a protein tag or a protein tag, specifically an affinity tag. Specific examples of the tag domain include His-tag (Hisx6), His-Strep-tag, strep-tag, avidin tag, flag™-tag, HA (hemagglutinin)-tag, T7-tag, V5-peptide-tag, GST (glutathione S-transferase)-tag, CBP (calmodulin-binding peptide)-tag, MBP (maltose-binding protein)-tag, and Myc-tag. When the binding partner is a molecule that exhibits specific binding to a target molecule, such as an antibody or an antigen-binding fragment thereof, or a derivative thereof, or an aptamer, the tag domain may be a peptide consisting of any amino acid sequence to which the molecule exhibiting specific binding can bind. When the binding partner described below is composed of a polypeptide, the tag domain in the fusion protein and the binding partner in the carrier may be used interchangeably.

[0043] The fusion protein may have one or more tag domains. In the latter case, the tag domain may be of one type or of multiple types. The tag domain may be located either at the N-terminus of the intein C or at the C-terminus of the intein N, or may be located at both.

[0044] The binding partner can be appropriately selected depending on the type of the tag domain. Specific examples of the binding partner include an antibody that recognizes the tag domain, an antigen-binding fragment thereof, or a derivative thereof; a nucleic acid molecule such as an aptamer; glutathione, calmodulin; a sugar chain such as mannose; a metal such as nickel, cobalt, or zinc, or an ion thereof; and the like.

[0045] The combination of the tag domain and the binding partner may be any combination that allows binding between the tag domain and the binding partner. Specifically, when the tag domain includes a His-tag, the binding partner may be, for example, nickel. When the tag domain includes a strep-tag or an avidin-tag, the binding partner may be, for example, biotin. When the tag domain includes an epitope tag such as a flag™-tag, an HA-tag, a T7-tag, a V5-peptide-tag, and / or a Myc-tag, the binding partner may be, for example, an antibody or antigen-binding fragment thereof against each epitope tag, or a derivative thereof, an aptamer, or the like. When the tag domain includes a GST-tag, the binding partner may be, for example, glutathione. When the tag domain includes a CBP-tag, the binding partner may be, for example, calmodulin. When the tag domain includes an MBP-tag, the binding partner may be, for example, mannose.

[0046] In the fusion protein, the target protein is not particularly limited and can be any protein to be cyclized and / or purified. Examples of the target protein include physiologically active proteins, pharmaceutical proteins, vaccine proteins, analytical proteins, and testing or diagnostic proteins. Examples of the target protein include antibodies such as monoclonal antibodies, multispecific antibodies, and antigen-binding fragments thereof; antibody derivatives such as single-chain fragments (scFvs) and tandem scFvs (e.g., BiTEs); cytokines, interferons, chemokines; peptide hormones such as insulin, growth hormone, and erythropoietin; enzymes such as trypsin, proteinase K, and collagenase; and viral antigens such as human papillomavirus, hepatitis B virus, and influenza virus. The antibody or antibody derivative is preferred. The activity of the tandem scFv can be enhanced by, for example, cyclization. Therefore, the tandem scFv is suitable as the target protein.

[0047] The fusion protein may contain one or more target proteins. In the latter case, the target proteins may be of one type or of multiple types.

[0048] In the fusion protein, the intein C and intein N that constitute the intein may be designed as an integrated intein, or a split intein may be used, for example.

[0049] Examples of the integrated intein include gp41-1, gp41-8, NrdJ-1, and SspGyrB. Examples of the split intein include DnaE (catalytic subunit α, dnaE-n, and dnaE-c of cyanobacterial DNA polymerase III), MCM2 (SEQ ID NO: 1, archaea Halorhabdus utahensis Examples include the Hut MCM-2 intein derived from DSM 12940, and a partial sequence of Uniprot: C7NUH7. In the amino acid sequence of SEQ ID NO: 1 below, the regions enclosed in brackets correspond, from the N-terminus, to intein N, the deleted region, and intein C (Reference 1). Reference 1: Ciragan A. et al., “Salt-inducible Protein Splicing in cis and trans by Inteins from Extremely Halophilic Archaea as a Novel Protein-Engineering Tool.” J Mol Biol. 2016 Nov 20;428(23):4573-4588. doi: 10.1016 / j.jmb.2016.10.006. Epub 2016 Oct 6. PMID: 27720988.

[0050] Amino acid sequence of MCM2 (SEQ ID NO: 1) [CVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATDG][GIAQIDTSSDRLGPAESGL][GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHN

[0051] The intein is preferably an intein derived from MCM2. When the intein is MCM2, examples of the MCM2-derived intein C and intein N include intein C consisting of the amino acid sequence of SEQ ID NO: 2 and intein N consisting of the amino acid sequence of SEQ ID NO: 3, respectively.

[0052] Amino acid sequence of MCM2-derived intein C (SEQ ID NO: 2) GIAQIDTSSDRLGPAESGL / GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHN

[0053] Amino acid sequence of MCM2-derived intein N (SEQ ID NO: 3) CVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATD

[0054] The intein is preferably an intein that undergoes a condition-dependent binding reaction (condition-dependent intein). The condition-dependent intein can also be referred to as an intein that undergoes a binding reaction between the intein and / or the intein C and intein N under certain conditions. Examples of the condition-dependent intein include salt-concentration-dependent inteins such as MCM2, a combination of HsaPolII and HsaCDC21 derived from Halobacterium salinarum NRC-1 (ATCC 700922) (see Reference 1), and temperature-dependent inteins (see Reference 2). The salt-concentration-dependent intein is, for example, an intein that undergoes a binding reaction between the intein and / or the intein C and intein N when the salt concentration is relatively high, i.e., the probability of the binding reaction occurring increases. The salt-concentration-dependent intein may also be an intein that undergoes a binding reaction when the salt concentration reaches a certain level. The salt-concentration-dependent intein is presumed to undergo a binding reaction that is dependent on the ionic strength of the salt. Therefore, the salt used to induce the salt-concentration-dependent intein binding reaction is not particularly limited, and any salt can be used. Examples of the salt include chlorides such as sodium chloride, calcium chloride, potassium chloride, and magnesium chloride; sulfates such as sodium sulfate, calcium sulfate, potassium sulfate, and magnesium sulfate; nitrates such as sodium nitrate, calcium nitrate, potassium nitrate, and magnesium nitrate; carbonates such as sodium carbonate, calcium carbonate, potassium carbonate, and magnesium carbonate; and phosphates such as sodium phosphate, calcium phosphate, potassium phosphate, and magnesium phosphate. When the salt-concentration-dependent intein is MCM2, the salt is preferably sodium chloride. Reference 2: Zeidler, M. et al. “Temperature-sensitive control of protein activity by conditionally splicing inteins.” Nat Biotechnol 22, 871-876 (2004). https: / / doi.org / 10.1038 / nbt979

[0055] The intein may be a functional equivalent to the extent that it maintains its protease and ligase activities. Examples of such functional equivalents include polypeptides that have protease and ligase activities and consist of an amino acid sequence that is 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the reference amino acid sequence of the intein. Examples of such functional equivalents include polypeptides that have protease and ligase activities and consist of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added to the reference amino acid sequence of the intein. The one or several amino acids may be, for example, 1 to 44, 1 to 33, 1 to 22, 1 to 11, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably a conservative substitution.

[0056] The number of inteins in the fusion protein may be one or more, but is preferably one because it allows for more accurate cyclization and purification of the target protein. When the fusion protein contains multiple inteins, the inteins may be one type or multiple types. The inteins are located at the N-terminus and C-terminus of the target protein.

[0057] The fusion protein preferably contains a soluble domain, for example, because this can improve the expression efficiency in the transformant. In the preparation step, animal cells or Escherichia coli ( Escherichia coli), the fusion protein can increase expression of the fusion protein by including the solubility domain. The solubility domain is preferably a polypeptide that, when fused to the fusion protein, increases the expression level of the fusion protein expressed in a transformant by at least 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 100% or more, compared to when the fusion protein is expressed as a fusion protein not containing the solubility domain. Examples of the solubility domain include proteins or partial polypeptides thereof. Specific examples of the solubility domain include GST, MBP, thioredoxin, antibodies, and antibody derivatives such as single-chain antibodies.

[0058] When the fusion protein contains the solubilization domain, the number of solubilization domains in the fusion protein may be one or more. In the latter case, the solubilization domain may be one type or multiple types. The solubilization domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N.

[0059] In the fusion protein, the intein C(I c ), the target protein (P t ), the intein N(I N ), and tag domain (T g ) can be arranged as shown in (1) or (2) below. (N-terminal) I c -P t -I N -T g (C-terminus)...(1) (N-terminus)T g -I c -P t -I N (C-terminus)...(2)

[0060] When the fusion protein contains the solubilization domain, the intein C(I c ), the target protein (P t ), the intein N(I N ), tag domain (T g ), and the solubilization domain (S) may be arranged, for example, as shown in (3) or (4) below. (N-terminus) SI c -P t -I N -T g (C-terminus)...(3) (N-terminus) T g -I c -P t -I N -S (C-terminus)...(4)

[0061] In the fusion protein, the intein C(I c ), the target protein (P t ), the intein N(I N ), tag domain (T g ), and the soluble domain (S) are directly or indirectly linked. The direct linkage means that the N- or C-terminal amino acid of a domain is bound to the C- or N-terminal amino acid of another domain via a peptide bond. On the other hand, the indirect linkage means that the N- or C-terminal amino acid of a domain is bound to the C- or N-terminal amino acid of another domain via a linker peptide (peptide linker). In other words, the N- or C-terminal amino acid of a domain is bound to the C- or N-terminal amino acid of the linker peptide via a peptide bond, and the amino acid at the other end of the linker peptide is bound to the N- or C-terminal amino acid of the other domain. The length of the linker peptide is, for example, 5 to 15 amino acids. The linker peptide may be a known linker peptide, and specific examples include a GS linker (GS, GGS, or GGGGS (SEQ ID NO: 7)), a linker peptide in which GS linkers are repeated ([GS] l , [GGS]m , or [GGGGS] n (l, m, and n are each an integer of 2 or greater), SEDEF (SEQ ID NO: 8), DKMR (SEQ ID NO: 9), GGGGSDKMR (SEQ ID NO: 10), etc. The linker of SEQ ID NO: 8 can be used, for example, as a linker between the intein C and the target protein. The linker of SEQ ID NO: 9 or 10 can be used as a linker between the target protein and the intein N.

[0062] The carrier may be any structure capable of supporting (e.g., immobilizing or retaining) the binding partner on its surface, and a common carrier can be used. Examples of the carrier include beads (particles) such as resin beads, agarose beads, magnetic beads, porous beads (porous bodies), and ionic (cationic or anionic) beads; tubes; columns containing such beads; and filters such as hollow fiber membranes, microfiltration membranes, ultrafiltration membranes, reverse osmosis membranes, and nonwoven fabrics. The carrier surface preferably has a hydrophilic surface. The hydrophilic surface can be formed, for example, by arranging hydrophilic functional groups such as carboxyl groups or amino groups on the carrier surface.

[0063] The binding partner is preferably supported on the carrier. In this case, the binding partner is directly or indirectly bound (e.g., immobilized or linked) to the carrier. In the case of direct binding, the bond between the binding partner and the carrier can be appropriately designed depending on the type of binding partner and the functional group on the surface of the carrier. As a specific example, if the binding partner is a protein and the carrier has a carboxyl group or an amino group as a functional group, the binding partner and the carrier form an amide bond to bind. In the case of indirect binding, the bond between the binding partner and the carrier can be appropriately designed depending on the type of binding partner and the functional group on the surface of the carrier. As a specific example, if the binding partner is a metal or a metal ion and the carrier has a chelating group as a functional group, the binding partner and the carrier are bound by forming a chelate between the metal or metal ion and the chelating group. The method of supporting the binding partner on the carrier can be appropriately set depending on, for example, the bond form between the binding partner and the carrier. The amount of the binding partner supported on the carrier is not particularly limited, and can be, for example, the same as the amount of the carrier and binding partner used in affinity chromatography.

[0064] In the retention step, the contact between the fusion protein and the carrier can be appropriately determined depending on the form of the fusion protein and the carrier. The contact is preferably performed in the presence of an aqueous solvent such as a buffer solution, physiological saline, or water. The fusion protein may be solid or liquid, but a liquid is preferred. When the fusion protein is a solid such as a powder, the fusion protein is preferably pre-dispersed in an aqueous solvent such as a buffer solution, physiological saline, or water. When the fusion protein is liquid, the fusion protein may be pre-diluted with an aqueous solvent such as a buffer solution, physiological saline, or water. As a specific example of the contact, when the fusion protein is a liquid containing the fusion protein (a fusion protein solution) and the carrier is beads carrying the binding partner, the contact can be performed, for example, by adding the beads to the fusion protein solution and then mixing the resulting solution. When the fusion protein is a fusion protein solution and the carrier is a column containing the beads, the contact can be performed, for example, by loading the fusion protein solution onto the column. In the retention step, solid-liquid separation may be performed after the contact by centrifugation, filtration, or the like.

[0065] In the holding step, when the fusion protein comes into contact with the carrier, the tag domain in the fusion protein binds to a binding partner on the carrier, and a complex between the fusion protein and the binding partner is formed on the carrier.

[0066] In the holding step, the contact and holding conditions may be the same or different, but the former is preferred from the viewpoint of ease of operation. The holding conditions may be any conditions that allow binding between the tag domain and the binding partner to occur, and can be appropriately set depending on the types of the tag domain and the binding partner.

[0067] The temperature in the holding step (holding temperature) is, for example, 0 to 30°C or 4 to 30°C.

[0068] The time period (holding time) in the holding step is, for example, 1 minute to 1 hour, 5 to 1 hour, or 10 minutes to 1 hour.

[0069] The pH in the holding step (holding pH) is, for example, pH 5 to 10, pH 6 to 9, or pH 6 to 8.5.

[0070] The contact and holding in the holding step are preferably performed under conditions that inactivate the enzymatic activity of the intein complex, i.e., under conditions (inactive conditions) that do not substantially cause a binding reaction of the intein complex. By carrying out the purification method of the present invention under such conditions, the yield of the circularized target protein can be improved, for example. The conditions under which substantially no binding reaction occurs are, for example, conditions in which the amount of circularized target protein is below the detection limit after contact and holding for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

[0071] When the intein is salt-dependent, the inactivation conditions are determined by the salt concentration. The salt concentration is, for example, the salt concentration in the fusion protein solution. Specific examples of inactivation conditions for the intein in MCM2 include a salt concentration of 250 mmol / L or less or 200 mmol / L or less during the contact and retention. The salt is, for example, sodium chloride (NaCl).

[0072] The purification method of the present invention may include, for example, a washing step of washing the carrier and the complex after the retention step. The purification method of the present invention can remove impurities (contaminants) other than the fusion protein from the carrier by washing, thereby purifying the circularized target protein with a higher purity. The washing method is not particularly limited and can be carried out, for example, by contacting the carrier carrying the fusion protein with a washing solution. In the washing method, for example, after contact with the washing solution, the carrier carrying the fusion protein may be separated from the washing solution. Specifically, when the carrier is beads, the washing can be carried out by, for example, adding the carrier carrying the fusion protein to the washing solution and then mixing the resulting solution. Furthermore, when the carrier is a column containing beads, the washing can be carried out by, for example, loading the washing solution onto the column.

[0073] The conditions for the washing step can be appropriately set depending on, for example, the holding and contact conditions in the holding step. The composition of the washing solution can be determined depending on, for example, the types of the tag domain and the binding partner. The composition of the washing solution is preferably one that does not affect the binding between the tag domain in the fusion protein and the binding partner on the carrier. Furthermore, the composition of the washing solution is preferably such that the enzymatic activity of the intein complex is inactive. The above explanation can be applied to the inactivation conditions. For example, a buffer such as a HEPES buffer or a Tris buffer can be used as the washing solution.

[0074] Next, in the separation step, the intein C and the intein N are bound to each other under conditions that allow a binding reaction between the intein C and the intein N to occur ( FIG. 1(D)). As a result, in the separation step, the circularized target protein can be separated from the intein C, the intein N, and the tag domain ( FIG. 1(E)). Furthermore, as shown in FIG. 1(E), in the purification method of the present invention, the target protein can be excised while each domain other than the target protein is supported on a carrier, allowing the circularized target protein to be purified. In the separation step, the circularized target protein is separated from the intein C, the intein N, and the tag domain that are supported on the carrier. Therefore, the separation step can also be referred to as a step of circularizing and separating the target protein from the carrier.

[0075] The separation step can be carried out, for example, by contacting the carrier on which the fusion protein is retained with a separation solution. Specifically, when the carrier is beads, the separation can be carried out, for example, by adding the carrier on which the fusion protein is retained to the wash solution and then mixing the resulting solution. Furthermore, when the carrier is a column containing beads, the separation can be carried out, for example, by loading the wash solution onto the column.

[0076] The temperature in the separation step (separation temperature) is, for example, 0 to 40°C, 4 to 37°C, or 4 to 30°C.

[0077] The time period in the separation step (separation time) is, for example, 1 minute to 48 hours, 30 minutes to 48 hours, or 1 to 48 hours.

[0078] The pH in the separation step (separation pH) is, for example, pH 5 to 10, pH 6 to 9, or pH 6.5 to 9.

[0079] The separation step is carried out under conditions (activation conditions) that allow a binding reaction between the intein C and intein N to occur. The activation conditions are not particularly limited and can be set appropriately depending on, for example, the types of intein C and intein N. The activation conditions are, for example, conditions that allow the target protein to be separated from 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the fusion protein retained on the carrier after a 30-minute separation treatment.

[0080] When the intein is salt-dependent, the activation conditions are determined by the salt concentration. The salt concentration is, for example, the salt concentration in the separation solution. Specific examples of activation conditions for the intein in MCM2 include a salt concentration in the separation solution of 0.5 to 5 mol / L, 0.75 to 4 mol / L, or 1 to 3.5 mol / L. The salt is, for example, sodium chloride (NaCl).

[0081] The composition of the separation solution is preferably such that it does not affect the binding between the tag domain in the fusion protein and the binding partner on the carrier.

[0082] The recovery step recovers the separated circularized target protein. In the purification method of the present invention, the circularized target protein separated in the separation step is contained, for example, in the separation solution used in the separation step. Therefore, in the purification method of the present invention, the recovery step may be carried out simultaneously with part or all of the separation step, or the recovery step may be carried out after the separation step.

[0083] The recovery step can be carried out, for example, by recovering the cyclized target protein that has separated and become free from the carrier. Specifically, the recovery step can be carried out by dispersing the cyclized target protein in a recovery solution and recovering the recovery solution after the dispersion. As a specific example of the recovery, when the carrier is beads carrying the binding partner, the recovery can be carried out, for example, by adding the beads to the recovery solution, mixing the resulting solution, and recovering the liquid fraction by solid-liquid separation using centrifugation, filter filtration, or the like. Furthermore, when the carrier is a column containing the beads, the recovery can be carried out, for example, by loading the recovery solution onto the column and recovering the eluate eluted from the column. In the recovery step, solid-liquid separation can be carried out during or after the recovery using centrifugation, filter filtration, or the like.

[0084] The conditions for the recovery step can be set, for example, in the same manner as those for the separation step, and the composition of the recovery liquid can be set, for example, in the same manner as the composition of the separation liquid.

[0085] In the recovery step, for example, the presence or absence of recovery of the target protein, its stability, and its activity in damaging cancer cells may be confirmed in accordance with the method in Example 1 described below.

[0086] The purification method of the present invention may include, for example, a purification step of purifying the circularized target protein after the recovery step. The purification method in the purification step can be, for example, a general protein purification method such as chromatography.

[0087] In this way, the purification method of the present invention can cyclize the target protein from the fusion protein and purify it. Furthermore, according to the purification method of the present invention, since the removal of the tag domain can be performed in conjunction with the purification of the target protein, the target protein can be purified after removing contaminants using the tag domain, and therefore the target protein can be purified with high purity.

[0088] <Method for producing target protein> In another aspect, the present invention provides a method for producing a cyclized target protein from a fusion protein containing the target protein. The production method of the present invention is a method for producing a cyclized target protein from a fusion protein containing the target protein, wherein the fusion protein comprises a tag domain, the target protein, and an intein, the intein comprising intein C and intein N, wherein in the fusion protein, the intein C is located at the N-terminus of the target protein and the intein N is located at the C-terminus of the target protein, and the tag domain is located at the N-terminus of intein C and / or the C-terminus of intein N. The production method of the present invention includes a step of cyclizing the target protein in the fusion protein, wherein the cyclization is carried out by the purification method of the present invention. The production method of the present invention is characterized in that the cyclization step is carried out by the purification method of the present invention, and other configurations and conditions are not particularly limited. The production method of the present invention can be performed using the explanations of the purification method, fusion protein, nucleic acid, expression vector, transformant, method for producing a transformant, and method for producing a fusion protein of the present invention.

[0089] <Fusion protein> In another aspect, the present invention provides a fusion protein that can be used for the circularization and purification of a target protein. The fusion protein of the present invention is a fusion protein for use in the circularization and purification of a target protein, comprising a tag domain, a target protein, and an intein, the intein comprising intein C and intein N, wherein intein C is located at the N-terminus of the target protein, intein N is located at the C-terminus of the target protein, the tag domain is located at the N-terminus of intein C and / or the C-terminus of intein N, and the intein is an intein that undergoes a condition-dependent binding reaction. The fusion protein of the present invention can be used to easily purify and produce the circularized target protein using the purification method or production method of the present invention. The fusion protein of the present invention can be used in conjunction with the explanations of the purification method, production method, nucleic acid, expression vector, transformant, method for producing a transformant, and method for producing a fusion protein of the present invention.

[0090] <Nucleic acid> In another aspect, the present invention provides a nucleic acid that can be used to synthesize a fusion protein. The nucleic acid of the present invention encodes the fusion protein of the present invention. The nucleic acid of the present invention is characterized by encoding the fusion protein of the present invention, and other configurations and conditions are not particularly limited. The nucleic acid of the present invention can be used to preferably produce the fusion protein of the present invention by genetic engineering techniques. The nucleic acid of the present invention can be used in conjunction with the explanations of the purification method, production method, fusion protein, expression vector, transformant, method for producing a transformant, and method for producing a fusion protein of the present invention.

[0091] The nucleic acid of the present invention can be designed by substituting corresponding codons based on the amino acid sequence of the fusion protein of the present invention. The base sequence of the nucleic acid of the present invention may be, for example, codon-optimized.

[0092] <Expression vector> In another aspect, the present invention provides an expression vector that can be used to synthesize a fusion protein. The expression vector of the present invention comprises the nucleic acid of the present invention. Using the expression vector of the present invention, the fusion protein of the present invention can be suitably produced by genetic engineering techniques. The explanations of the purification method, production method, fusion protein, nucleic acid, transformant, method for producing a transformant, and method for producing a fusion protein of the present invention can be used for the expression vector of the present invention.

[0093] The expression vector of the present invention is, for example, an expression vector into which the nucleic acid of the present invention is inserted. The expression vector means, for example, a nucleic acid molecule that can transport an inserted gene into a target such as a cell.

[0094] The expression vector is not particularly limited in its configuration, as long as it contains a polynucleotide encoding a fusion protein so that the fusion protein encoded by the polynucleotide of the nucleic acid of the present invention can be expressed.

[0095] The expression vector can be prepared, for example, by inserting a polynucleotide encoding a fusion protein, i.e., the nucleic acid of the present invention, into a backbone vector (hereinafter also referred to as a "basic vector"). The type of the expression vector is not particularly limited and can be appropriately determined depending on, for example, the type of the host.

[0096] Examples of the host include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells. Examples of the prokaryotic organism include Escherichia coli ( Escherichia coli ) and other Escherichia species, Pseudomonas putida ( Pseudomonas putida Examples of the eukaryote include bacteria of the genus Pseudomonas, such as Saccharomyces cerevisiae ( Saccharomyces cerevisiae Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.

[0097] Examples of the expression vector (basic vector) include viral vectors and non-viral vectors. When the heat shock method is used to transform a host, the expression vector may be, for example, a binary vector. Examples of the expression vector include pETDuet-1, pQE-80L, and pUCP26Km. When bacteria such as Escherichia coli are transformed, examples of the expression vector include pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, and pUC8. When yeast is transformed, examples of the expression vector include pYepSec1, pMFa, and pYES2. When insect cells are transformed, examples of the expression vector include pAc and pVL. When transforming mammalian cells, examples of the expression vector include pCDM8 and pMT2PC.

[0098] The expression vector preferably has a regulatory sequence that regulates the expression of the polynucleotide encoding the fusion protein and the expression of the fusion protein of the present invention encoded by the polynucleotide of the fusion protein. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the expression vector is not particularly limited. In the expression vector, the regulatory sequence may be located in any manner that allows it to functionally regulate the expression of the polynucleotide encoding the fusion protein and the expression of the fusion protein encoded thereby, and can be located according to known methods. For example, the regulatory sequence may utilize a sequence already contained in the basic vector, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence contained in the basic vector may be replaced with another regulatory sequence.

[0099] The expression vector may further comprise, for example, a coding sequence for a selection marker, such as a drug resistance marker, a fluorescent protein marker, an enzyme marker, or a cell surface receptor marker.

[0100] Insertion of DNA, insertion of the regulatory sequence, and / or insertion of the coding sequence of the selection marker into the expression vector may be carried out, for example, by a method using restriction enzymes and ligase, or by using a commercially available kit, etc.

[0101] <Transformants and methods for producing transformants> In another aspect, the present invention provides a transformant capable of producing a fusion protein and a method for producing the same. The transformant of the present invention comprises a nucleic acid encoding the fusion protein of the present invention. The transformant of the present invention is characterized by comprising a nucleic acid encoding the fusion protein of the present invention, and other configurations and conditions are not particularly limited. The transformant of the present invention can suitably produce the fusion protein of the present invention.

[0102] The method for producing a transformant of the present invention includes the step of introducing the nucleic acid of the present invention into a host. The method for producing a transformant of the present invention is characterized by introducing the nucleic acid of the present invention into the host, and other steps and conditions are not particularly limited. The transformant can be produced by the method for producing a transformant of the present invention. The explanations for the purification method, production method, fusion protein, nucleic acid, expression vector, and production method of fusion protein of the present invention can be cited for the transformant and its production method of the present invention.

[0103] In the transformant of the present invention, the nucleic acid encoding the fusion protein can be the same as that described above for the nucleic acid encoding the fusion protein of the present invention. The nucleic acid of the present invention may be the expression vector of the present invention.

[0104] In the transformant of the present invention, the nucleic acid of the present invention is present as an exogenous molecule. Therefore, the transformant of the present invention can be produced, for example, by introducing the nucleic acid of the present invention into the host.

[0105] The method for introducing the nucleic acid is not particularly limited and can be performed using known methods. The nucleic acid may be introduced, for example, using the expression vector. The introduction method can be appropriately selected depending on, for example, the type of host. Examples of the introduction method include introduction using a gene gun such as a particle gun, the calcium phosphate method, the polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid introduction, DEAE-dextran method, direct injection using microglass tubes, the hydrodynamic method, the cationic liposome method, methods using introduction adjuvants, and Agrobacterium-mediated methods. Examples of liposomes include lipofectamine and cationic liposomes, and examples of introduction adjuvants include atelocollagen, nanoparticles, and polymers. When the host is a microorganism, a method mediated by, for example, E. coli or Ps. putida is preferred. The polynucleotide of the fusion protein of the present invention may be introduced into the host using, for example, the expression vector of the present invention.

[0106] <Method of producing fusion protein> In another aspect, the present invention provides a method for producing a fusion protein. The method for producing a fusion protein of the present invention includes an expression step of expressing the fusion protein of the present invention. The method for producing a fusion protein of the present invention is characterized by including the expression step, and other steps and conditions are not particularly limited. The fusion protein of the present invention can be produced by the method for producing a fusion protein of the present invention. The explanations for the purification method, production method, fusion protein, nucleic acid, expression vector, transformant, and method for producing the transformant of the present invention can be used for the method for producing a fusion protein of the present invention.

[0107] The fusion protein may be expressed, for example, using the expression vector of the present invention. The method for expressing the fusion protein is not particularly limited, and known methods can be used, for example, a host or a cell-free protein synthesis system.

[0108] In the former case, it is preferable to use a host into which the fusion protein or a nucleic acid containing the fusion protein has been introduced, and to express the fusion protein in the host by culturing the host. In this way, for example, by introducing the fusion protein or a nucleic acid containing the fusion protein into a host, a transformant that synthesizes the fusion protein of the present invention can be produced, and the fusion protein can be synthesized by culturing the transformant.

[0109] The method for culturing the host is not particularly limited and can be appropriately determined depending on the type of the host. The medium used for culturing is not particularly limited and can be appropriately determined depending on the type of the host.

[0110] In the latter case, it is preferable to express the polynucleotide of the fusion protein in a cell-free protein synthesis system. In this case, an expression vector may be used to express the polynucleotide of the fusion protein. The cell-free protein synthesis system can be carried out by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which a polynucleotide encoding the fusion protein has been introduced, and for example, a commercially available reagent kit can be used.

[0111] The method for producing a fusion protein of the present invention may include, for example, a recovery step of recovering the fusion protein. The fusion protein obtained in the recovery step may be, for example, a crude product or a purified protein.

[0112] When the protein is recovered from the culture medium, in the recovery step, insoluble matter is removed, for example, by filtering the culture supernatant, centrifuging the culture supernatant, etc. Then, in the recovery step, the fusion protein can be obtained by separating and purifying the culture supernatant after the removal of insoluble matter, for example, by an appropriate combination of concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; dialysis; and chromatography using various columns such as an ion exchange column and a gel filtration column. Cut.

[0113] When the fusion protein is recovered from the transformant, the recovery step involves disrupting the transformant by, for example, pressure treatment, ultrasonic treatment, etc. Then, the resulting disruption solution is subjected to removal of insoluble matter, separation, and purification as described above to obtain the fusion protein.

[0114] The fusion protein obtained by the production method of the present invention may be used, for example, as a crude enzyme (unpurified enzyme) as it is, or as a partially purified enzyme, or as a single purified enzyme.

[0115] In the production method of the present invention, the obtained fusion protein may be powdered by, for example, freeze-drying, vacuum drying, spray drying, etc. In this case, in the production method of the present invention, for example, the fusion protein may be dissolved in advance in a buffer solution such as acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD buffer (e.g., PIPES, MES, MOPS, etc.). [Example]

[0116] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples.

[0117] [Example 1] It was confirmed that the tag peptide of the fusion protein of the present invention can be removed in conjunction with the purification of the target protein by the purification method of the present invention.

[0118] (1) Preparation of recombinant Cyclic Ex3 fusion protein The full-length synthetic gene containing the base sequences of MCM2 intein C (SEQ ID NO: 2), MCM2 intein N (SEQ ID NO: 3), VHH (SEQ ID NO: 4, solubility domain), and Ex3 (BiTE) (SEQ ID NO: 5, target protein) was synthesized by PCR. Ex3 is a tandem scFv consisting of an anti-CD3 single-chain antibody and an anti-EGFR single-chain antibody linked via a linker peptide. The amino acid sequence enclosed in brackets in the amino acid sequence of SEQ ID NO: 5 below contains, from the N-terminus, the Ia1 VH domain, hOKT3 VH domain, hOKT3 VL domain, h528 VH domain, and h528 VL domain, in that order. This full-length gene was ligated into an animal cell expression vector (pCAGEN) to construct a recombinant protein expression vector. The expression vector was constructed by linking, in order from the N-terminus to the C-terminus, the secretory signal sequence-VHH, MCM2 intein C, Ex3 (BiTE), MCM2 intein N, and a His tag (SEQ ID NO: 6 below). Next, the recombinant protein expression vector was transfected into Expi293F cells. The cells were cultured in HE400 medium at 37°C in a CO2 incubator for 7 days, and the recombinant protein was expressed in the medium supernatant.

[0119] Amino acid sequence of VHH (SEQ ID NO: 4) QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS

[0120] Ex3 (BiTE) (SEQ ID NO: 5) [QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSS]AGGGG SGGGGSGGGGS[DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQIT]RAGGGGS [QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNIWPGSGGTNYAEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSGGPYFFDYWGQGTLVTVSS]AGGGGS GGGGSGGGGS[DIVMTQSPLSLPVTPGEPASISCRSSQNIVHNNGITYLEWYLQKPGQSPQLLIYKVSDRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPPTFGQGTKVEIK]AA

[0121] Cyclic Ex3 fusion protein (SEQ ID NO: 6) [MEFGLSWLFLVAILKGVQC][QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHNSED]SEDEF[QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSS]AGGGGSGGGGSGGGGS[DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQIT]RAGGGGS[QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNIWPGSGGTNYAEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSGGPYFFDYWGQGTLVTVSS]AGGGGSGGGGSGGGGS[DIVMTQSPLSLPVTPGEPASISCRSSQNIVHNNGITYLEWYLQKPGQSPQLLIYKVSDRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPPTFGQGTKVEIK]AADKMR[DKMRCVTGDTLVQAGDGRRRIRELAGETAEAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATDG]HHHHHH

[0122] Next, the suspension after the animal cell culture was collected and centrifuged to recover the culture supernatant. After the collection, the target protein was purified. Specifically, imidazole and sodium chloride were added to the supernatant (120 mL) to final concentrations of 10 mmol / L and 150 mmol / L, respectively. The protein was loaded onto a 500 μL Ni-NTA column for adsorption. After washing with 10 mL of washing buffer (50 mmol / L Tris-HCl (pH 8.0), 10 mmol / L imidazole, 150 mmol / L NaCl), the column was eluted with 5 mL of elution buffer (50 mmol / L Tris-HCl (pH 8.0), 500 mmol / L imidazole, 150 mmol / L NaCl) and dialyzed against PBS buffer.

[0123] To the purified protein (protein concentration: 1.08 μmol / L, 200 μL, 150 mmol / L NaCl, 50 mmol / L HEPES), 100 μL of Ni-NTA resin was added and suspended. After suspension, the mixture was centrifuged at 6000 rpm for 10 minutes. After centrifugation, the supernatant (i) was removed. After removal, 200 μL of washing buffer (500 mmol / L NaCl, 50 mmol / L Tris-HCl, 10 mmol / L imidazole, pH 8.0) was added and suspended. After suspension, the mixture was centrifuged at 6000 rpm for 10 minutes. After centrifugation, the supernatant (ii) was removed. After the removal, 200 μl of reaction buffer (3 mol / l NaCl, 0.5 mmol / l TCEP, 50 mol / l HEPES, 10 mM imidazole, pH 7.0) was added, and the mixture was shaken at 37° C. for 24 hours. After the shaking, the mixture was centrifuged at 6000 rpm for 10 minutes to obtain supernatant (iii).

[0124] (2) Study of target protein elution The purified protein was used to examine whether the target protein, cyclized Ex3 (BiTE), could be ligated on a Ni-NTA column and eluted. Specifically, SDS-PAGE was used to examine whether cyclized Ex3 (BiTE) was eluted in the reaction buffer after centrifugation. 200 μl of each sample was precipitated with TCA, and 50 μl of SDS-PAGE sample buffer was added to the precipitate after centrifugation and suspended. The suspension was then heated at 95°C for 5 minutes. After the heat treatment, 20 μl of the sample was applied to the wells of a 10% polyacrylamide gel and electrophoresed. The application order was from left to right on the gel: marker, purified protein, supernatant (i), supernatant (ii), supernatant (iii), eluted solution, and eluted Ni-NTA resin. After obtaining the supernatant (iii), 200 μl of elution buffer (500 mmol / L NaCl, 500 mmol / L imidazole, 50 mmol / L Tris-HCl (pH 8.0)) was added and suspended. After suspension, the suspension was centrifuged at 6000 rpm for 10 minutes. The supernatant after centrifugation was used as the eluted solution, and the precipitate after centrifugation was used as the eluted Ni-NTA resin. These results are shown in Figure 2.

[0125] Figure 2 shows photographs of proteins at each step of purification. The top of the photograph indicates the sample type, and the left side indicates the molecular weight (kDa). In Figure 2, the lanes, from left to right, show marker (M), purified protein (P), supernatant (i) (FT), supernatant (ii) (W), supernatant (iii) (Cyclic EX3BiTE), eluted solution (E), and Ni-NTA resin (Ni resin) after elution. As shown in Figure 2, a band representing cyclized Ex3(BiTE) was detected in supernatant (iii) after addition of reaction buffer and centrifugation (boxed area). In contrast, no band representing cyclized Ex3(BiTE) was detected in the other samples. These results demonstrate that the cyclized target protein Ex3(BiTE) is separated in the presence of reaction buffer, which is the condition for the binding reaction between intein C and intein N. This separation is suggested to occur through the binding of intein C and intein N.

[0126] (3) Preparation of recombinant Ex3 ta6 protein A recombinant Ex3 ta6 protein was prepared as a control for comparison with the cyclized Ex3 target protein obtained by the method of the present invention. Specifically, pROXb3-Ex3ta6 was transformed into Brevibacillus competent cells (HPD31-sp3 strain). After transformation, the resulting colonies were inoculated into 2 ml of 2SL (4%) medium (Nm(+); fc 50 μg / ml). Preculture was performed at 120 rpm and 30°C for 24 hours. After preculture, 800 ml of 2SL medium (Nm(+); fc 50 μg / ml, L-(+)-arginine; fc 0.2 mol / l) was inoculated with 1% of the preculture solution. Subsequently, main culture was performed at 120 rpm and 30°C for 48 hours. After main culture, the resulting culture solution was centrifuged at 4500 × g and 4°C for 20 minutes. The supernatant was recovered. The protein in the supernatant was concentrated using ammonium sulfate. Prior to the concentration, the ammonium sulfate particles were broken down using a pestle. The supernatant was then stirred with a stirrer bar in a cold room (4°C) without creating bubbles. The stirring was performed while gradually adding ammonium sulfate equivalent to 60% of the supernatant's mass. After stirring for 1 hour, the mixture was centrifuged at 4500 × g and 4°C for 20 minutes. After the centrifugation, the supernatant was removed. Approximately 4 ml of 1 × PBS was added to the precipitate to dissolve it. After the dissolution, the resulting solution was dialyzed three times using 1 × PBS (first time: 1 hour, second time: 2 hours, third time: overnight) to remove residual ammonium sulfate. After the three dialysis cycles, the solution was centrifuged at 15000 × g and 4°C for 15 minutes. After the centrifugation, the supernatant was recovered. The supernatant was purified using a column packed with 1 ml of Ni Sepharose. The elution in the purification was performed by increasing the imidazole concentration in 1x PBS to 0 mmol / L, 10 mmol / L, 50 mmol / L, 150 mmol / L, 200 mmol / L, 300 mmol / L, and 500 mmol / L, respectively, at 50 CV, 100 CV, 20 CV, 10 CV, 10 CV, 10 CV, and 10 CV. After the elution, the purity of the eluted fractions was confirmed using SDS-PAGE.After this confirmation, the eluate in which the Ex3 ta6 band was clearly observed was concentrated by ultrafiltration. After this concentration, the Ex3 ta6 monomer fraction was fractionated using a Superdex 200 Increase 10 / 300 GL column. After this fractionation, calibration was performed using the Gel Filtration Calibration Kit LMW and the Gel Filtration Calibration Kit HMW. SDS-PAGE was performed on the peak near the molecular weight of Ex3 ta6 to evaluate whether Ex3 ta6 had been purified. After this evaluation, the fraction containing the target antibody was concentrated by ultrafiltration. After this concentration, the resulting concentrate was filter-sterilized to obtain the Ex3 ta6 recombinant protein.

[0127] Ex3 ta6 (SEQ ID NO: 11) QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSAGGGGSGGGS GGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRAGGGGSQVQLVQSGAEVKK PGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNIWPGSGGTNYAEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSGGPYFFDYWGQGTLVTVSSAGGGGSGGGGGSGGGGSDIVMTQSPLS LPVTPGEPASISCRSSQNIVHNNGITYLEWYLQKPGQSPQLLIYKVSDRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPPTFGQGTKVEIKAAAAEQKLISEEDLNLGGGMRGGSHHHHHH

[0128] (4) Examination of the thermal stability of the target protein The thermostability of the target protein, cyclized Ex3, obtained by the method of the present invention was examined. Specifically, the 0.050 mg / ml Ex3 ta6 solution obtained in Example 1(3) and the supernatant (iii) obtained in Example 1(1) after addition of the reaction buffer and centrifugation were adjusted to 0.050 mg / ml using the reaction buffer to obtain each protein solution. SYPRO Orange (S5692, Sigma) was diluted 50-fold with deionized water. 5 μl of SYPRO Orange was added to the Ex3 ta6 solution and the protein solution and mixed. After mixing, each mixture was heated from 30°C to 100°C at a rate of 0.5°C / 30 seconds using a real-time PCR system (Mx3000P, Stratagene) and fluorescence was measured at each temperature. Fluorescence measurements were performed using an excitation wavelength of 492 nm and an emission wavelength of 580 nm. These results are shown in Figure 3.

[0129] Figure 3 is a graph showing the thermal stability of cyclized Ex3. In Figure 3, the horizontal axis represents temperature, and the vertical axis represents the value obtained by differentiating fluorescence intensity with respect to temperature. As shown in Figure 3, the denaturation temperature of cyclized Ex3 obtained by the method of the present invention was 60.5°C, and the denaturation temperature of Ex3 ta6 was 59.5°C. These results demonstrate that the thermal stability of cyclized Ex3 obtained by the method of the present invention is equivalent to that of Ex3 ta6.

[0130] (5) Examination of the cancer cell damage activity of target proteins We investigated whether cyclized Ex3, the target protein obtained by the method of the present invention, induces cancer cell cytotoxicity. Specifically, the MTS assay was used to measure the cancer cell cytotoxicity. The culture supernatant of statically cultured TFK-1 cells (a cell line derived from human extrahepatic cholangiocarcinoma) was aspirated, followed by the addition of 1 ml of TrypLE™ Express Enzyme (1×), no phenol red (Gibco). The cells were then allowed to stand at 5% CO2 and 37°C for 5 minutes. After the addition, the flask was gently tapped. After the tapping, 9 ml of 1× PBS was added, and the cells were detached from the flask by suspension to obtain a cell suspension. The cell suspension was then centrifuged at 300 × g for 5 minutes. After the centrifugation, the supernatant was aspirated, and 10 ml of RPMI1640 medium containing 10% FBS and penicillin / streptomycin was added and suspended to obtain a cell suspension. Thereafter, 10 μl of the cell suspension was stained with 10 μl of trypan blue (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the number of cells was counted using a cell counter. 5 The cell suspension was taken and the medium was added to the cell suspension so that the volume of the medium became 12 ml, and the cell suspension was obtained. After the suspension, 5.0 × 10 cells were placed in each well of a 96-well plate. 3 100 μL of the cell suspension was seeded per well to give cells / 100 μL. After seeding, the cells were incubated under conditions of 5% CO2, 37°C, and 24 hours. After incubation, the supernatant was removed by suction. Next, 2.5 × 10 cells were seeded per well. 4T-LAK cells (activated lymphocytes) were seeded at 50 μl per cell, or 100 μl of ultrapure water or RPMI 1640 medium was added. As in Example 1(4), the supernatant (iii) obtained in Example 1(1) after addition of reaction buffer and centrifugation was diluted with the reaction buffer to obtain protein solutions of different concentrations (Cyclic Ex3). Furthermore, supernatants containing Clyclic Ex3 G1 (SEQ ID NO: 12), in which a GGGGS (SEQ ID NO: 7) linker was inserted at the linking site of each domain, were obtained in the same manner as in Example 1(1), and diluted with the reaction buffer to obtain protein solutions of different concentrations (Clyclic Ex3 G1). Ex3 ta6 obtained in Example 1(3) was used as a control. The concentrations were adjusted to five levels: 0.01 pmol / L, 0.1 pmol / L, 1 pmol / L, 10 pmol / L, and 100 pmol / L. 50 μl of the protein solution with different concentrations was added to each well seeded with the T-LAK cells. After the addition, the plate was incubated under conditions of 5% CO2 and 37°C for 24 hours. After the incubation, the supernatant was aspirated. After the aspirated removal, the wells were washed three times with 1×PBS. Next, 1 ml of MTS reagent (CellTiter 96™ Aqueous One Solution Cell Proliferation Assay) was diluted by adding it to 11 ml of RPMI 1640 medium. The diluted MTS reagent was added to the wells at 100 μl / well. After the addition, the plate was incubated under conditions of 5% CO2 and 37°C for 40 minutes to 1 hour. After the incubation, the absorbance at 490 nm (reference: 655 nm) was measured using a plate reader (Thermo Fisher Scientific). The incubation was continued until the absorbance of the wells containing only RPMI 1640 medium reached approximately 0.8. The detected absorbance was then used to calculate the growth inhibition rate (%) of cancer cells. The growth inhibition rate (%) of cancer cells was calculated as follows: (1-(average absorbance of wells to which the protein solution was added-average absorbance of wells to which ultrapure water was added)) / (average absorbance of wells to which RPMI1640 medium was added-average absorbance of wells to which ultrapure water was added) x 100. The results are shown in Figure 4.

[0131] Clyclic Ex3 G1 (SEQ ID NO: 12) [MEFGLSWLFLVAILKGVQC][QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQMN SLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQGTQVTVSS][GDIGLRTIENVESVPDHDYDSVYDLTVEGTHNFLANGMVVHNSED]EF[QVQLVQSGGG VVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVT VSS][AGGGGSGGGGSGGGGS][DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIA TYYCQQWSSNPFTFGQGTKLQIT]RAGGGGS[QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNIWPGSGGTNYAEKFKNRVTMTRDT SISTAYMELSRLRSDDTAVYYCARSGGPYFFDYWGQGTLVTVSS]AGGGGSGGGGSGGGGS[DIVMTQSPLSLPVTPGEPASISCRSSQNIVHNNGITYLEWYLQK PGQSPQLLIYKVSDRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPPTFGQGTKVEIK]AAGGGGSDKMR[CVTGDTLVQAGDGRRRIRELAGETA EAGSIEELPNGRTIRDVDIDVWTMTDDETLTRRPVTAIHEYDAPETLYEVTLSTGEEVTVTPDHPFFIEQASGRVETPAEDLQPGDLVFVPEGSAMATDG]HHHHHH

[0132] Figure 4. Cyclization of Ex3 in vitro4 is a graph showing the cytotoxic activity against cancer cells in each of the cyclized Ex3s obtained by the method of the present invention. In FIG. 4, the horizontal axis represents the concentration of the protein solution, and the vertical axis represents the growth inhibition rate (%) against cancer cells. As shown in FIG. 4, cyclized Ex3 (Cyclic Ex3 and Clyclic Ex3 G1) exhibited a higher growth inhibition rate than non-cyclized Ex3 ta6, i.e., a higher cytotoxic activity against cancer cells. Furthermore, it was found that the growth inhibitory activity of Clyclic Ex3 G1 against cancer cells was higher than that of cyclized Ex3. These results demonstrate that the growth inhibitory activity of cyclized Ex3 obtained by the method of the present invention against cancer cells is higher than that of Ex3 ta6, indicating a high growth inhibitory activity against cancer cells.

[0133] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0134] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Purification method> (Appendix 1) 1. A method for circularizing and purifying a target protein from a fusion protein comprising the target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; contacting the fusion protein with a carrier comprising a binding partner capable of binding to the tag domain, and allowing the tag domain to bind to the binding partner to form a complex, thereby retaining the fusion protein on the carrier; allowing the intein C and the intein N to bind to each other under conditions that allow a binding reaction between the intein C and the intein N to occur, and separating the circularized target protein from the intein C, the intein N, and the tag domain; and recovering the separated circularized target protein. (Appendix 2) The method according to Appendix 1, wherein the intein is an intein that undergoes a condition-dependent binding reaction. (Appendix 3) 3. The method of claim 2, wherein the condition is salt concentration. (Appendix 4) The method according to any one of Appendices 1 to 3, wherein the intein is an intein in which a binding reaction between the intein C and the intein N occurs when the salt concentration becomes relatively high. (Appendix 5) 5. The method according to any one of Appendices 1 to 4, wherein the condition for the binding reaction between the intein C and the intein N is a salt concentration of 1 mol / L or more. (Appendix 6) 6. The method of any one of claims 1 to 5, wherein the intein is a split intein. (Appendix 7) 7. The method of any one of claims 1 to 6, wherein the intein is MCM2. (Appendix 8) The intein C comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 8. The method of any one of claims 1 to 7, wherein the intein N comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. (Appendix 9) the fusion protein comprises a solubilization domain; 9. The method of any one of claims 1 to 8, wherein the solubility domain is located N-terminally of the intein C. (Appendix 10) 10. The method of claim 9, wherein the solubility domain comprises a protein. (Appendix 11) 11. The method of any one of claims 1 to 10, wherein the target protein comprises an antibody or an antigen-binding fragment thereof. (Appendix 12) 12. The method of any one of claims 1 to 11, wherein the target protein is a multispecific antibody. (Appendix 13) 13. The method according to any one of claims 1 to 12, comprising the step of contacting a sample containing the fusion protein with a carrier containing a binding partner capable of binding to the tag domain, and allowing the tag domain to bind to the binding partner to form a complex, thereby retaining the fusion protein on the carrier. (Appendix 14) 14. The method of claim 13, wherein the sample is a protein preparation. (Appendix 15) 15. The method of any one of claims 1 to 14, comprising expressing the fusion protein prior to said retaining. (Appendix 16) 16. The method of any of appendix 1 to 15, wherein the tag domain is selected from the group consisting of His-tag, His-Strep-tag, strep-tag, flag-tag, HA-tag, T7-tag, V5-peptide-tag, GST-tag, CBP-tag, MBP-tag, and Myc-tag. <Manufacturing method> (Appendix 17) 1. A method for producing a circularized target protein from a fusion protein containing the target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; circularizing the target protein in the fusion protein; 17. The method of claim 1, wherein the cyclization is carried out by the method of any of claims 1 to 16. <Fusion protein> (Appendix 18) 1. A fusion protein for use in circularizing and purifying a target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; The intein is a fusion protein in which a binding reaction occurs conditionally. (Appendix 19) 19. The fusion protein of claim 18, wherein the condition is salt concentration. (Appendix 20) 20. The fusion protein of claim 18 or 19, wherein the intein is MCM2. (Appendix 21) The intein C comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 21. A fusion protein according to any one of claims 18 to 20, wherein the intein N comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. (Appendix 22) the fusion protein comprises a solubilization domain; 22. The fusion protein of any one of claims 18 to 21, wherein the solubility domain is located N-terminal to the intein C. (Appendix 23) 23. The fusion protein of claim 22, wherein the solubility domain comprises a protein. (Appendix 24) 24. The fusion protein of any of claims 18 to 23, wherein the target protein comprises an antibody or an antigen-binding fragment thereof. (Appendix 25) 25. The fusion protein of any of claims 18 to 24, wherein the target protein is a multispecific antibody. <Nucleic acid> (Appendix 26) A nucleic acid encoding a fusion protein for use in circularizing and purifying a target protein according to any one of appendices 18 to 25. <Transformants> (Appendix 27) A transformant comprising the nucleic acid described in Appendix 26. <Method of producing a transformant> (Appendix 28) A method for producing a transformant, comprising the step of introducing the nucleic acid according to Appendix 26 into a host. <Method of producing fusion protein> (Appendix 29) A method for producing a fusion protein, comprising the steps of culturing the transformant according to Appendix 27 and expressing the fusion protein. [Industrial Applicability]

[0135] As described above, according to the present invention, tag peptide removal from a fusion protein containing a target protein can be performed in conjunction with the purification of the target protein. Furthermore, according to the present invention, the circularized fusion protein maintains the activity and stability of the protein, and is therefore useful, for example, for purifying physiologically active proteins. Therefore, the present invention is extremely useful, for example, in the pharmaceutical field.

Claims

1. 1. A method for circularizing and purifying a target protein from a fusion protein comprising the target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; contacting the fusion protein with a carrier comprising a binding partner capable of binding to the tag domain, and allowing the tag domain to bind to the binding partner to form a complex, thereby retaining the fusion protein on the carrier; allowing the intein C and the intein N to bind to each other under conditions that allow a binding reaction between the intein C and the intein N to occur, and separating the circularized target protein from the intein C, the intein N, and the tag domain; and recovering the separated circularized target protein; The method, wherein the intein is an intein in which a binding reaction occurs conditionally.

2. The method of claim 1 , wherein the condition is salt concentration.

3. The method according to claim 1 or 2, wherein the condition for the binding reaction between the intein C and the intein N is a salt concentration of 1 mol / L or more.

4. The method of any one of claims 1 to 3, wherein the intein is MCM2.

5. the fusion protein comprises a solubilization domain; The method of any one of claims 1 to 4, wherein the solubility domain is located on the N-terminal side of the intein C.

6. 6. The method of claim 1, wherein the target protein comprises an antibody or an antigen-binding fragment thereof.

7. 1. A method for producing a circularized and purified target protein from a fusion protein comprising the target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; Circularizing and purifying the target protein in the fusion protein, 7. A method, wherein the circularization and purification is carried out by the method of any one of claims 1 to 6.

8. 1. A fusion protein for use in circularizing and purifying a target protein, comprising: the fusion protein comprises a tag domain, a target protein, and an intein; The intein includes intein C and intein N, In the fusion protein, the intein C is located on the N-terminus of the target protein; the intein N is located on the C-terminus of the target protein; the tag domain is located on the N-terminal side of the intein C and / or the C-terminal side of the intein N; The intein is a fusion protein in which a binding reaction occurs conditionally.

9. The fusion protein of claim 8 , wherein the condition is salt concentration.

10. The fusion protein of claim 8 or 9, wherein the intein is MCM2.

11. the fusion protein comprises a solubilization domain; The fusion protein of claim 8 , wherein the solubility domain is located N-terminally of the intein C.

12. 12. The fusion protein of claim 8, wherein the target protein comprises an antibody or an antigen-binding fragment thereof.

Images