JP2010075090A - Protein expression vector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an expression vector efficiently expressing a protein the expression of which has been difficult by a conventional vector: to provide a transformant obtained by introducing the expression vector: and to provide a method or the like for producing the protein by using the expression vector. <P>SOLUTION: The expression vector for expressing the target protein contains a DNA encoding the whole or a part of a transcription factor ATF2 (Activating Transcription Factor 2) protein, and an insertion site for the insertion of a DNA encoding the target protein so that the target protein may be expressed as a fused protein with the whole or the part of the ATF2 protein. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention uses an expression vector capable of efficiently expressing a protein that has been difficult to express by conventional recombinant protein expression methods, a transformant obtained by introducing the expression vector, and the expression vector. The present invention relates to a protein production method and the like.

A system for efficiently expressing various foreign proteins in bacteria such as E. coli has been established. Proteins expressed using these systems are desired to be soluble and active proteins for their applications.
However, depending on the protein to be expressed, if its higher-order structure formation is not successful and becomes insoluble, a large amount of abnormal proteins with different three-dimensional structures may be produced due to folding abnormality, or it may be hardly soluble or aggregated in the first place. In some cases, it is difficult to solubilize the protein. These insoluble proteins and abnormal proteins are known to exist as aggregates called inclusion bodies in the host cell.

In order to increase the solubility of a protein to be expressed, a method for expressing the protein as a fusion protein with various proteins (tags) has been developed. For example, it is known that a thioredoxin tag can be used to produce a larger amount of soluble protein than usual (see, for example, Patent Document 1). A method using glutathione S transferase (GST) as a fusion tag is also known (see, for example, Patent Document 2). However, even if these methods are used, there are many genes that are difficult to express and proteins that are produced only in a trace amount.
Moreover, when an inclusion body is formed, the expressed fusion protein can be obtained by solubilizing and purifying the fusion protein contained in the inclusion body. However, purification often requires an antibody that can recognize this tag efficiently. It is not easy to find a tag in which an antibody that enhances the solubility of the fusion protein and can be recognized efficiently exists.

  As a method for efficiently purifying an expressed protein, a method of fusing a histidine tag for protein expression is also known. The histidine tag fusion protein expressed by this method can be purified by affinity chromatography using affinity for several metals such as nickel. Then, the target protein can be separated from the thus purified histidine fusion protein using a protease. However, even in this method, if the target protein has a cleavage site for protease, the fusion protein may interfere with the binding with nickel, or the purification conditions may affect the activity, but it is not necessarily functional. No (Patent Document 3).

In addition, for the purpose of detecting or purifying the expressed fusion protein, an antibody against the non-target protein of the fusion protein may be used. However, there are few excellent antibodies that recognize the protein, and there is a problem that sufficient detection, purification, construction of applied technology, etc. may be difficult.
For this reason, a vector capable of sufficiently obtaining a protein difficult to obtain with a conventional expression vector has been desired.

Furthermore, with conventional expression vectors, it has often been difficult to express a polypeptide. For example, a polypeptide consisting of about 100 residues, which is a partial sequence of a protein, may be used as an immunizing antigen. However, the conventional expression system often does not apply to conditions under which such a polypeptide can be expressed. For this reason, there was a problem that a polypeptide having a sufficient amount and property for immunization could not be obtained.
Japanese Patent No. 2513978 JP-T-1-503441 Japanese National Patent Publication No. 11-503617

  An object of the present invention is obtained by introducing an expression vector that can efficiently express a protein that has been difficult to express with conventional vectors and that can be easily purified, and the expression vector. It is to provide a transformant, a protein production method using the expression vector, and the like.

  As a result of intensive studies to solve the above problems, the present inventors have used a vector containing DNA encoding all or part of the transcription factor ATF2 (activating transcription factor 2, hereinafter referred to as “ATF2”) protein. The present inventors have found that, by expressing a fusion protein of the target protein and all or part of the transcription factor ATF2 protein, it is possible to efficiently express a protein that was difficult to express with a conventional vector. It came to be completed.

That is, the present invention includes the following inventions.
[1] An expression vector for expressing a target protein, the DNA encoding all or part of the transcription factor ATF2 (Activating transcription factor 2) protein, and the target protein comprising all or part of the ATF2 protein An expression vector comprising an insertion site for inserting a DNA encoding a target protein so that the protein is expressed as a fusion protein;
[2] The expression vector according to [1] above, wherein the target protein and all or part of the ATF2 protein are expressed as a fusion protein by inserting a DNA encoding the target protein;
[3] The expression vector according to [2] above, further comprising DNA encoding a linker peptide that connects all or part of the ATF2 protein and the target protein in the fusion protein;
[4] The expression vector according to [3] above, wherein the DNA encoding the linker peptide consists of the base sequence set forth in SEQ ID NO: 13, 14, or 39;
[5] The expression vector according to any one of [1] to [4], further comprising DNA encoding a purification tag protein;
[6] The expression vector according to [5] above, wherein the DNA encoding the purification tag protein has the base sequence set forth in SEQ ID NO: 15 or 16.
[7] The DNA according to any one of [1] to [6] above, wherein the DNA encoding all or part of the ATF2 protein is a DNA comprising the base sequence set forth in any one of SEQ ID NOs: 1 to 6. The described expression vector;
[8] Expression vector deposited as pBMR (deposit number NITE P-522);
[9] The expression vector according to any one of [1] to [8], further comprising DNA encoding the target protein;
[10] A transformant obtained by introducing the expression vector according to [9] above;
[11] DNA comprising the base sequence according to any one of SEQ ID NOs: 1 to 6;
[12] a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 7 to 12;
[13] An antibody that binds to the polypeptide of [12] above;
[14] A hybridoma cell line deposited as clone number ATF2-00158 (deposit number FERM P-21570);
[15] The antibody according to [13] produced by the cell line according to [14] above;
[16] A method for producing a target protein comprising the following steps (a) to (c):
(A) a step of culturing the transformant according to claim 10 in a medium (b) a step of collecting the fusion protein produced in the culture by the step (a) from the culture, and a step (c) ( c) cleaving the fusion protein collected in b) into all or part of the ATF2 protein and the target protein [17] In the step (b), by column or immunoprecipitation using an antibody against the ATF2 protein, The method according to [16] above, wherein the fusion protein is collected;
[18] A method for producing a target protein comprising the following steps (d) to (f):
(D) a step of culturing the transformant according to claim 10 in a medium (e) a fusion protein produced in the culture by the step (d), comprising all or part of the ATF2 protein, a target protein, And (f) a step of purifying the target protein obtained by the step (e) [19] In the step (f), all or part of the ATF2 protein obtained in the step (e) The target protein is purified by removing all or part of the ATF2 protein from the mixture with the target protein by column or immunoprecipitation using an antibody against the ATF2 protein. Method;
[20] A method for producing a fusion protein comprising the following steps (g) to (h):
(G) a step of culturing the transformant according to claim 10 in a medium, and (h) a step of collecting the fusion protein produced in the culture by the step (g) from the culture [21] In the step (h), the fusion protein is collected by a column or immunoprecipitation using an antibody against the ATF2 protein;
[22] The method according to [17], [19] or [21] above, wherein the antibody is the antibody according to [13] or [15] above;
[23] A method for detecting a fusion protein of all or part of a transcription factor ATF2 protein and a target protein, the transcription factor ATF2 contained in the fusion protein using the antibody according to [13] or [15] above A method comprising the step of recognizing all or part of the protein;
[24] A method of purifying a fusion protein of all or part of the transcription factor ATF2 protein and the target protein, the transcription factor contained in the fusion protein using the antibody according to [13] or [15] above Recognizing all or part of the ATF2 protein and purifying the fusion protein;
[25] DNA encoding a fusion protein comprising all or part of the transcription factor ATF2 protein and the target protein;
[26] The DNA according to [25] above, which comprises a linker DNA between DNA encoding all or part of the ATF2 protein and DNA encoding the target protein;
[27] The DNA according to [26] above, wherein the linker DNA consists of the base sequence described in SEQ ID NO: 13, 14, or 39;
[28] The DNA according to any one of [25] to [27] above, which contains a DNA encoding a purification tag protein at the carboxy terminus of the DNA encoding the target protein;
[29] The DNA according to [28] above, wherein the DNA encoding the purification tag protein comprises the base sequence described in SEQ ID NO: 15 or 16;
[30] The DNA according to any one of [25] to [29] above, wherein the whole or part of the sequence of the ATF2 protein consists of the base sequence according to any of SEQ ID NOs: 1 to 6;
[31] A fusion protein comprising all or part of the transcription factor ATF2 protein and a target protein.

  By using the expression vector of the present invention, a protein that has been difficult to express can be expressed efficiently. Furthermore, according to the expression vector of the present invention, the target protein can be obtained as a fusion protein with all or part of the ATF2 protein, and thus can be easily purified using an anti-ATF2 protein antibody. According to the expression vector of the present invention, it is possible to produce a large amount of a recombinant protein for the purpose of treatment, diagnosis or other research use.

The “expression vector” of the present invention is an expression vector for expressing a desired target protein, which is an expression vector containing DNA encoding all or part of the transcription factor ATF2 protein, and transcription factor ATF2 Any expression vector is included as long as it contains DNA encoding all or part of the protein. The “expression vector” of the present invention efficiently expresses a fusion protein of the target protein and all or part of the transcription factor ATF2 protein by inserting a DNA encoding the target protein.
Therefore, the expression vector of the present invention has an insertion site for inserting a DNA encoding the target protein so that all or part of the transcription factor ATF2 protein and the target protein can be expressed as a fusion protein. The insertion site can be a restriction site that is cleaved by various restriction enzymes.

  ATF2 used in the expression vector of the present invention is known as a DNA binding protein. The ATF2 gene encodes a transcription factor that is a protein of the leucine zipper family of DNA binding proteins. ATF2 protein binds to cAMP response element (CRE), which is an eight-base palindromic structure. This protein forms a homodimer or heterodimer with c-Jun to stimulate CRE-dependent transcription. This protein is also known to be a histone acetyltransferase (HAT) that specifically acetylates histones H2B and H4 in vitro.

  Among the “all or part of the transcription factor ATF2 protein” of the present invention, “the whole transcription factor ATF2 protein” is a protein having substantially the same amino acid sequence as the human transcription factor ATF2 protein represented by SEQ ID NO: 38. In addition, a protein comprising an amino acid sequence having a homology of at least 70%, preferably 80%, more preferably 95% or more is expressed efficiently when expressed as a fusion protein with the target protein. . In addition, as long as such an effect is exhibited, a part of the amino acid sequence shown in SEQ ID NO: 38 is deleted, a part of which is substituted with another amino acid, and another amino acid is added or inserted. Also included.

Further, among “all or part of the transcription factor ATF2 protein” of the present invention, the “part of the transcription factor ATF2 protein” of the present invention may be any as long as it includes a part of the ATF2 protein. Is a partial sequence of 1 to 200 amino acid residues, for example, preferably 10 to 150 bases long, more preferably 15 bases to 129 bases long. The “part of the transcription factor ATF2 protein” of the present invention also has an effect of efficiently expressing the target protein when expressed as a fusion protein with the target protein.
As the “part of transcription factor ATF2 protein” of the present invention, for example, the 226th amino acid of the ATF2 full-length sequence (SEQ ID NO: 38) showing “the whole human transcription factor ATF2 protein” in FIG. In the case of the first number, ATF2 tag position numbers 1 to 129 (SEQ ID NO: 7), 29th to 129th (SEQ ID NO: 8), 51st to 129th (SEQ ID NO: 9) of the amino acid sequence 95 to 129 (where 95 is methionine) (SEQ ID NO: 10), 29th to 95th (SEQ ID NO: 11), 29th to 50th (SEQ ID NO: 12) are desirable. These peptides were designed by the present inventors with reference to the epitope sequence contained in the partial sequence of ATF2. Hereinafter, the peptides consisting of the amino acid sequences described in SEQ ID NOs: 7 to 12 may be referred to as “ATF2 partial peptides 1 to 6”, respectively.

Examples of the “DNA encoding all or part of the transcription factor ATF2 protein” contained in the expression vector of the present invention include, for example, the base sequence encoding the protein described in SEQ ID NO: 38 and the partial sequences of SEQ ID NOS: 7 to 12 And DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 6, which all express the human transcription factor ATF2 protein as a fusion protein with the target protein. Hereinafter, DNAs comprising a base sequence encoding a part of the ATF2 protein described in SEQ ID NOs: 1 to 6 may be referred to as “ATF2 partial DNAs 1 to 6”, respectively.
Furthermore, “DNA encoding all or part of the transcription factor ATF2 protein” of the present invention includes DNA having high homology with DNA encoding all or part of the transcription factor ATF2 protein of the present invention. For example, DNA that hybridizes under highly stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6 can be mentioned. Such DNA has a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more with the base sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6. Examples thereof include DNA having a base sequence possessed. In addition, as the “DNA encoding all or part of the transcription factor ATF2 protein” of the present invention, those derived from humans are preferably used. For example, in a heterologous species to the human transcription factor ATF2 protein, All or part of homologous genes, family gene groups, mutant genes and the like are also included.
As used herein, highly stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, the sodium concentration is 10 mM to 300 mM, preferably 20 mM to 100 mM, and the temperature is 25 ° C to 70 ° C, preferably 42 ° C to 55 ° C. Hybridization can be performed by a known method, for example, annealing by adjusting the environmental temperature.

The expression vector of the present invention is not particularly limited as long as it is an expression vector containing DNA encoding all or part of the transcription factor ATF2 protein. For example, any of plasmid DNA, phage DNA and the like may be used.
As the plasmid DNA, for example, when the host cell into which the expression vector of the present invention is introduced is E. coli, pET vector (Novagen), pRSET vector (Invitrogen), pCYB vector (NEW ENGLAMD), which is a vector for E. coli. Bio Labs), pBR vector, pUC vector and the like. When the host cell is yeast, the yeast vector pESP-1 expression vector (STRATAGENE), pAUR123 vector (TAKARA), pYES2 vector (Invitrogen), YEp13 vector, YEp24 vector, YCp50 vector, etc. Is mentioned. When the host cell is Bacillus subtilis, examples of the vector for Bacillus subtilis include pUB110 vector and pTP5 vector. Examples of phage DNA include λ phage.
Furthermore, when animal cells are used as hosts, animal viruses such as retroviruses or vaccinia viruses, and insect virus vectors such as baculoviruses can also be used. Examples thereof include a pMAM-neo expression vector (CLONTECH), a pCDNA4 / TO vector (Invitrogen), a pBK-CMV vector (STRATAGENE) which are vectors for animal cells. In addition, when the host cell is an insect cell, pBacPAK vector (manufactured by CLONTECH), pAcUW31 vector (manufactured by CLONTECH), pAcP (+) IE1 vector (manufactured by Novagen) and the like, which are vectors for insect cells, can be mentioned.

The expression vector of the present invention may contain a replication origin, a selection marker, a promoter, etc. in addition to DNA encoding all or part of the transcription factor ATF2 protein, and if necessary, an enhancer, terminator, ribosome binding site, polyadenyl Linking signal, linker DNA, etc.
As the replication origin contained in the expression vector of the present invention, for example, those derived from ColE1, R factor, and F factor can be used for the E. coli vector. For the yeast vector, for example, 2 μm DNA derived from ARS1 can be used. For animal cell vectors, for example, SV40 and adenovirus-derived vectors can be used. Moreover, in order to make it possible to use any bacterium, the expression vector of the present invention may be constructed so as to include a plurality of replication origins.

  As the promoter contained in the vector of the present invention, trp promoter, lac promoter, PL promoter, PR promoter and the like can be used for E. coli vectors. For yeast vectors, gal1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like can be used. For animal cell vectors, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like can be used.

  As a selection marker contained in the vector of the present invention, a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, and the like can be used for an E. coli vector. For yeast vectors, Leu2, Trp1, Ura3 genes and the like can be used. Neomycin resistance gene, thymidine kinase gene, dihydrofolate reductase gene and the like can be used for animal cell vectors.

The expression vector of the present invention can also incorporate a DNA encoding a tag protein for detection and purification of the fusion protein.
The “purification tag protein” of the present invention refers to a tag used for detection and purification of a fusion protein or a target protein, and a protein for which detection or purification methods have been established is preferable. Examples of DNA encoding the tag protein for purification include, for example, glutathione-S-transferase, maltose-binding protein, histidine (His6), DNA encoding histidine-glycine-histidine sequence, and the nucleotide sequence set forth in SEQ ID NO: 15 or 16 DNA consisting of

  In the present invention, the “fusion protein” of all or part of the ATF2 protein and the target protein is a transcription of DAN encoding all or part of the ATF2 protein and DNA encoding the target protein by one promoter. A protein obtained from RNA. Here, all or a part of the ATF2 protein and the target protein may be directly bonded or may be bonded via a linker peptide.

As the linker peptide, those that can be selectively cleaved or digested by chemical or enzymatic methods known to those skilled in the art can be used. For example, a peptide containing methionine, a peptide containing Asn-Gly Then, it can be cleaved with cyanogen bromide, hydroxylamine or the like. It can also be a peptide (peptide encoded by SEQ ID NO: 39) having a sequence enzymatically cleaved by a proteolytic enzyme such as trypsin, enterokinase, factor Xa, collagenase and thrombin. A peptide encoded by the base sequence described in SEQ ID NO: 13 or 14 is also preferably used.
When a linker peptide is used, a linker DNA is placed between the DNA encoding all or part of the ATF2 protein and the DNA encoding the target protein in the expression vector of the present invention. “Linker DNA” refers to an oligodeoxynucleotide inserted into the binding site of two DNA fragments and encodes a linker peptide.

The linker DNA of the present invention can be used for purposes other than providing a cleavage site. For example, the linker peptide may be a simple amino acid sequence that is long enough to prevent steric hindrance between the protein of interest and the rest of the fusion protein.
Whether or not the linker DNA is necessary depends on the structural characteristics of the target protein and whether or not the produced fusion protein is useful even if it is not cleaved. Therefore, the DNA encoding the fusion protein of the present invention may not contain linker DNA. For example, the DNA sequence encoding the target protein and the DNA sequence encoding all or part of the transcription factor ATF2 protein may be directly fused at the amino or carboxyl terminus.

  Examples of the expression vector of the present invention as described above also include an expression vector deposited as pBMR (deposit number NITE P-522) containing DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2. pBMR includes a DNA (SEQ ID NO: 39) encoding a thrombin digestion site as a linker inserted downstream of a partial sequence of the ATF2 protein, and further includes a DNA (GTGTCCGTG) encoding His-Gly-His as a purification tag protein. It is a vector.

Furthermore, the present invention includes those in which the above-described expression vector contains DNA encoding the target protein.
The “target protein” of the present invention refers to a protein to be expressed, and means an arbitrary amino acid chain regardless of its length or post-transcriptional modification. The “target protein” of the present invention includes polypeptides and peptide fragments, and may be any of natural, synthetic or recombinant.
For example, even if it is a protein which cannot be collect | recovered with a normal expression system, the protein which can be made into the object of expression in this invention is mentioned. For example, zinc finger protein 143 (ZNF143), metastasis associated 1 family, member 2 (MTA2), nuclear receptor subfamily 4 group A member 1 (NR4A1), signal transducer and transcriptional activator 3 (STAT3), high mobility group AT-hook 2 protein (HMGA2), KIN, EMG1, nuclear factor I / B (NFIB) and the like are suitably expressed as target proteins by the vector of the present invention.

  The “transformant” of the present invention refers to a transformant obtained by introducing the expression vector of the present invention containing a DNA encoding a target protein into a host. The host is not particularly limited as long as it can express the fusion protein gene. For example, bacteria belonging to the genus Escherichia such as Escherichia coli, Bacillus genus such as Bacillus subtilis, Pseudomonas putida such as Pseudomonas putida; Saccharomyces cerevisiae, -Yeast such as Pombe (Schizosaccharomyces pombe); Animal cells such as monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, human GH3, human FL cells; or insect cells such as Sf9, Sf21 Is mentioned.

  The expression vector can be introduced into the host by a known method depending on the type of the host. Examples thereof include a heat shock method, a calcium phosphate method, an electroporation method, a spheroplast method, a lithium acetate method, a lipofection method, a cation lipopolyamine method, a liposome method, and an Agrobacterium method. Moreover, gene transfer into each of the above host cells can be performed by a method not using a recombinant vector, such as a particle gun method.

  The “polypeptide” of the present invention refers to a polypeptide that is all or part of the transcription factor ATF2 sequence, and examples thereof include a polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 7 to 12. In addition to polypeptides having substantially the same amino acid sequence as these polypeptides, including those that are at least 70%, preferably 80%, more preferably 95% or more homologous to this polypeptide . Also included are those in which a part of the partial sequence of transcription factor ATF2 is deleted, a part of which is substituted with another amino acid, and another amino acid added or inserted.

  The “antibody” of the present invention refers to an antibody that recognizes the polypeptide described in any one of SEQ ID NOs: 7 to 12 which is a partial sequence of the human transcription factor ATF2 sequence, and may be either a polyclonal antibody or a monoclonal antibody. Also included is a monoclonal antibody produced by the cell line deposited under clone number ATF2-00158 (deposit number FERM P-21570). The monoclonal antibody is a monoclonal antibody having ATF2 partial peptide 1 as an antigen.

  The antibody of the present invention can be produced by any conventionally known production method. The monoclonal antibody can be produced by the usual hybridoma technique using the whole or part of the transcription factor ATF2 protein, preferably the polypeptide described in any one of SEQ ID NOs: 7 to 12 as an antigen. Polyclonal antibodies can be produced by conventional methods such as inoculating a host animal such as a mouse or rat with the protein of the present invention and recovering serum.

  The “hybridoma cell line” of the present invention refers to a cell line capable of producing an antibody that recognizes all or part of the protein of the transcription factor ATF2 sequence, and deposited as clone number ATF2-00158 (deposit number FERM P-21570). Including

The “method for producing a target protein” of the present invention comprises (a) a step of culturing the transformant of the present invention in a medium, (b) a step of collecting a fusion protein produced thereby from the culture, c) A step of separating the collected fusion protein into all or part of the transcription factor ATF2 protein and the target protein.
Here, in the step (b), the fusion protein can be collected by a column using an antibody against the ATF2 protein or by immunoprecipitation.
Further, another embodiment of the “method for producing a target protein” of the present invention includes (d) a step of culturing the transformant of the present invention in a medium, (e) a fusion protein produced thereby, and ATF2 protein. And a step of separating the protein of interest into a target protein, and (f) a step of purifying the obtained protein of interest.
Here, in step (e), a mixture of all or part of the ATF2 protein and the target protein is obtained. In step (f), the mixture is obtained, for example, by a column using an antibody against ATF2 protein or by immunoprecipitation. The target protein can be purified by removing the ATF2 protein from the protein.
The “method for producing a fusion protein” of the present invention comprises the steps of (g) culturing the transformant of the present invention in a medium, and (h) collecting the fusion protein produced thereby from the culture.
In step (h), the fusion protein can be collected by column or immunoprecipitation using an antibody against ATF2 protein as in step (b).

Cultivation of the transformant of the present invention (step (a), (d) or (g)) can be performed according to a usual method used for culturing the transformant.
As a culture medium, when cultivating a transformant using microorganisms such as Escherichia coli and yeast as host cells, the medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism, and can be cultured efficiently. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Any carbon source may be used as long as the organism can assimilate, and carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, tryptone, meat extract, corn steep liquor, etc. It is done. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

The culture is usually performed at 16 to 37 ° C for about 6 to 62 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH may be maintained at 7.0 to 7.5. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium depending on the vector selection marker.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using a Lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added to the medium. In addition, when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacetic acid (IAA) or the like may be added to the medium.
As a medium for culturing a transformant obtained using animal cells as a host, generally used RPMI1640 medium, DMEM medium, a medium obtained by adding fetal calf serum or the like to these mediums, or the like is used. The culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During the culture, antibiotics such as kanamycin and penicillin may be added to the medium according to the vector selection marker.

After culturing, the fusion protein is produced in the culture. The “culture” of the present invention refers to a medium or a host cell.
Collection from the culture of the fusion protein of the present invention (step (b)) can be performed according to a commonly used method. For example, when the fusion protein is produced in the cells or cells, the fusion protein is extracted by crushing the cells or cells. When the fusion protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like.
After the step (b), a step of purifying the fusion protein may be performed before cleaving all or part of the ATF2 protein and the target protein.
For purification of the fusion protein, any method known to those skilled in the art may be used. By using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, the fusion protein can be extracted from the culture. Can be isolated and purified.
For example, the fusion protein can be recovered by binding to the carrier with the tag (histidine or the like) and eluting with imidazole to release the fusion protein from the carrier.

  The antibody of the present invention may be used for purification of the fusion protein. For example, there are methods of purifying the fusion protein by an antibody column using a monoclonal antibody against the transcription factor ATF2 protein or by immunoprecipitation. In addition, an antibody against the target protein or purification tag can also be used. The method for producing the antibody column is not particularly limited, and the monoclonal antibody of the present invention may be directly or indirectly immobilized on a solid support by a usual method. As the immunoprecipitation method, any method known to those skilled in the art may be used. For example, an antigen (for example, a fusion protein), an antibody, or a secondary antibody-conjugated sepharose bead (for example, Sepharose 4B) can be used. Further, instead of Sepharose beads, in addition to magnetic beads, Sepharose, agarose, etc., those in which protein A, protein G, etc. are coupled to a carrier such as magnetic beads, Sepharose, agarose, etc. can also be used.

Any method known to those skilled in the art may be used to cleave all or part of the transcription factor ATF2 protein of the present invention and the target protein. The fusion protein of the present invention can be cleaved, for example, at a linker peptide site inserted between all or part of the transcription factor ATF2 protein of the present invention and the target protein. Further, only the target protein can be released from the carrier by digesting with a protease while the fusion protein is bound to the carrier.
In the method for producing a target protein according to the present invention, the target protein may be purified after cleaving the fusion protein into all or part of the ATF2 protein and the target protein.
Cleavage of all or part of the ATF2 protein and the target protein can be performed according to the method described above, and any method known to those skilled in the art may be used for purification of the target protein. After cleavage, a method for purifying the target protein from a mixture of all or part of the ATF2 protein and the target protein is a general biochemical method used for protein isolation and purification, such as ammonium sulfate precipitation, gel filtration, Ion exchange chromatography, affinity chromatography and the like can be used alone or in appropriate combination.
For example, all or part of the ATF2 protein can be removed from the mixture by an antibody column using a monoclonal antibody against the transcription factor ATF2 protein according to the present invention or by immunoprecipitation. The antibody column production method and immunoprecipitation method can be performed according to the methods described.
Any method known to those skilled in the art may be used as a method for detecting a fusion protein or a target protein using the antibody of the present invention. For example, enzyme immunoassay (such as ELISA and EIA), fluorescent labeling immunoassay, radioimmunoassay (radioimmunoassay, RIA), luminescence immunoassay, immunoturbidimetric method, latex agglutination method, latex turbidimetric method, red blood cell Examples include agglutination, particle agglutination, Western blotting, and surface plasmon resonance.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Construction of expression vector according to the present invention)
1. Removal of His • tag of pET21b (Novagen Cat No.69016-3) and introduction of nucleotide sequence encoding His-Gly-His sequence pET21b in combination with primer Pst21F (SEQ ID NO: 17) and primer Xho21R (SEQ ID NO: 18) PCR was carried out using as a template. 5 μL of buffer solution, 5 μL of dNTP, 2 μL of magnesium sulfate, 1 μL of template DNA, 15 pmol of each primer solution and 1 μL of Taq polymerase were added, and water was added to make a total volume of 50 μL. After heating at 94 ° C. for 2 minutes, PCR was performed under the condition that this step was repeated 40 cycles, with one cycle consisting of cooling at 50 ° C. for 30 seconds and heat retention at 68 ° C. for 30 seconds. After confirming the amplification of the 4303-139 region-CTCGAG (XhoI site) (about 1.1 kb) of the pET21b vector map by electrophoresis, it was digested with restriction enzymes PstI and XhoI (37 ° C., 5 hours). This digested product was ligated with pET21b (159-4308 region) digested with restriction enzymes PstI and XhoI using DNA ligase. This prepares a plasmid from which the 140-157 region of pET21b, ie, 18 bases encoding the His.tag sequence; CACCACCACCACCACCAC (SEQ ID NO: 37) has been deleted (plasmid A).

2. Introduction of base sequence encoding His-Gly-His sequence PCR was carried out using pET21b as a template with a combination of primer Hind21F (SEQ ID NO: 19) and primer Mlu21R (SEQ ID NO: 20). After confirming the amplification of the 166-1069 region (about 0.9 kb) of the pET21b vector map with bases added in the order of 9 bases (GTGTCCGTG) encoding XhoI site-His-Gly-His, the restriction enzyme MluI and Digested with XhoI (37 ° C., 5 hours). Ligation was performed using plasmid A digested with restriction enzymes MluI and XhoI and DNA ligase so that the digested product functions as a purification tag.
This plasmid was transformed into Escherichia coli DH5α to prepare a plasmid from a positive clone, which was designated as pET21bHGH. pET21bHGH was digested with NdeI and BamHI (37 ° C., 5 hours).

3. Introduction of nucleotide sequence encoding ATF2
Oligonucleotide (SEQ ID NO: 21) and 3 'designed to add an NdeI site (CATATG) to the 5' end of ATF2 partial DNA 1 (DNA encoding the 226-354 region of Homo sapiens Activating transcription factor 2 (ATF2)) PCR was performed using the partial DNA1 as a template, using the oligonucleotide SEQ ID NO: 22 designed to add at the end the base sequence encoding the thrombin digestion site consisting of amino acids LVPRGS as a linker and the BamHI site (GGATCC).
The product obtained by PCR was digested with NdeI and BamHI (37 ° C., 5 hours). And it mixed with pET21bHGH obtained previously and ligated using DNA ligase.

4). Construction of an expression vector according to the present invention The thus prepared plasmid was transformed into Escherichia coli DH5α strain to obtain a colony. These colonies were grown on LB medium + Amp50, and the plasmid was extracted by the alkaline SDS method. The base sequence of this plasmid was confirmed with a DNA sequencer, and the expression vector according to the present invention was constructed.

(Expression of human transcription factors MTA2, NR4A1, NFIB and PAX8)
As a DNA encoding a target protein to be introduced downstream of ATF2 partial DNA 1, a partial sequence of human transcription factor MTA2 gene (SEQ ID NO: 23), a partial sequence of human transcription factor NR4A1 gene that was found to be insoluble (SEQ ID NO: 24) In addition, partial sequences of human transcription factors NFIB and PAX8 genes (SEQ ID NOs: 25 and 26, respectively) were used. Expression of these four types of target proteins (peptides encoded by the nucleotide sequences described in SEQ ID NOs: 23 to 26) as soluble proteins is recognized by a Thioredoxin reductase (hereinafter referred to as TRX) tag.

DNAs encoding these target proteins were amplified by PCR, and as shown in FIG. 1, using the BamHI and HindIII sites added to the 5 ′ end and 3 ′ end, respectively, the ATF2 partial DNA 1 of the prepared plasmid vector The DNA was ligated downstream using DNA ligase.
The success or failure of the introduction of the sequence was determined by nucleotide sequence analysis. The expression plasmid thus obtained was transformed into Escherichia coli Rosetta-gami ^™ (DE3) to prepare a transformant.
On the other hand, as a comparative example, an expression plasmid in which the DNAs described in SEQ ID NOs: 23 to 26 are inserted into the multi-cloning site of pET21b, or an expression plasmid in which DNAs described in SEQ ID NOs: 23 and 24 are inserted by adding a TRX tag The transformant having the plasmid was cultured in 1.5 mL of LB + Amp50 medium for 9-12 hours (preculture), and 200 μL of this culture solution was added to 10 mL of LB + Amp50 medium (2% inoculation) Culture was performed at 37 ° C. for 4 hours (note that when pET21b was used, the target protein was expressed as a fusion protein with a T7 tag (11 residues) derived from pET21b).
Four hours after the start of culture, isopropyl β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM to induce expression. After addition of IPTG, the cells were further cultured for 3 hours (37 ° C.) to complete the culture (culture method 1).
For the preparation of the expressed protein, the culture solution of E. coli was collected by centrifugation at 5000 rpm, 4 ° C. for 10 minutes, and washed with sterilized water or the like. As a control experiment, a transformant that does not express a foreign protein (Rosetta-gami ^™ (DE3) / pET21b) was added to the experimental system. These cells were suspended in 500 μL of a buffer solution for cell disruption (8 M urea, 100 mM sodium dihydrogen phosphate, 10 mM Tris-HCl (pH 8.0)), and the whole protein was extracted by shaking and stirring for 1 hour. Cell disruption with an ultrasonic crusher was performed as necessary. Thereafter, the suspension was separated into a solution portion and a residue portion by centrifugation at 15000 rpm, 4 ° C. for 15 minutes, the protein concentration of the solution portion was calculated, and SDS-PAGE was performed under the condition of 5 μg / lane (DRC Perfect) NT Gel 15-25%, electrophoresis conditions 500V, 50mA, 60min).
As a result, when ATF2 partial DNA 1 was added, expression of all target proteins was observed (FIG. 2). In the two types of partial sequences, results equivalent to or higher than those obtained when the TRX tag was added were found, indicating the effectiveness of the vector according to the present invention. When the T7 tag was added, some of the target protein expression was observed (lanes 1 and 9), but some were not observed (lanes 4 and 7).

Table 1 shows the proteins indicated by the arrows in each lane of FIG.

(Construction of plasmid vector and confirmation of protein expression)
The ATF2 partial peptides 1 to 6 designed with reference to the epitope sequence contained in the partial sequence of ATF2 were examined for the influence on the expression of the target protein.
As a control, a TRX tag was used.
In the examination, the partial DNA sequence of NR4A1 described above was fused downstream of the ATF2 partial DNA or TRX tag. Transformants of Escherichia coli Rosetta-gami ^™ (DE3) were obtained using the expression plasmids prepared for each. The expression of each fusion protein was confirmed by SDS-PAGE according to culture method 1. As a result, promotion of expression was observed in all (FIG. 3). Among them, a vector (pBMR) expressing ATF partial peptide 2 was used in the following experiment.

Table 2 shows the proteins indicated by the arrows in each lane of FIG.

(Confirmation of protein expression)
Expression Study Using the ATF2 partial peptide 2 examined previously, expression with respect to various peptide sequences (DNAs encoding the peptides shown in the lower table of FIG. 4 are shown in SEQ ID NOs: 27 to 36 and 40) was evaluated. As the method, culture method 1 and SDS-PAGE were used.
The results are summarized in FIG. 4 together with the results of Example 2. The expression was confirmed regardless of differences in chain length and isoelectric point, and the origin species, and it was found that the target protein that could not be solubilized with the conventional tag (TRX) might be solubilized.

(Confirmation of antibody recognition for expressed protein)
A monoclonal antibody recognizing this sequence has been obtained for the ATF2 partial peptide (monoclonal antibody produced by a cell line deposited under the deposit number FERM P-21570. Hereinafter referred to as “anti-ATF2 antibody” in the Examples) Sometimes this antibody.) Thus, the NR4A1 sequence was fused downstream of the six types of ATF2 partial peptides or TRX tags described in Example 3, and fusion protein was prepared and detected by Western blotting.

The method was performed as follows.
(Preparation of fusion protein)
1 g of wet cells were suspended in the cell disruption buffer described in Example 2 and disrupted by shaking for 60 minutes. The residue was removed by centrifugation (48300 G, 4 ° C., 15 minutes) to obtain a supernatant. The supernatant was adsorbed to a nickel chelate resin through a purification tag added to the fusion protein, and then eluted with an acidic pH. The obtained elution fraction was dialyzed with phosphate buffered saline containing 1.75 M urea to prepare a sample.
(Western blotting)
The concentration of each fusion protein was adjusted to 40 to 500 ng / lane and SDS-PAGE was performed. Using a methanol-activated PVDF membrane soaked in Transfer buffer (4.79 mM Tris, 3.86 mM glycine, 0.128 mM SDS) for 10 minutes with a BIORAD TRANS-BLOT SD SEMI-DRY TRANSFER CELL Transfer to PVDF membrane at 14V, maximum ampere, 120 minutes.
The transferred PVDF membrane was equilibrated by shaking for 10 minutes with TBS-T (2.48 mM Tris-HCl, 137 mM sodium chloride, 0.27 mM potassium chloride, 0.05% Tween20 (pH 7.4)). TBS- containing 5% skim milk Blocked with T (30 minutes to 1 hour).
After that, PVDF was thoroughly washed with TBS-T and anti-ATF2 antibody (Biomatrix Laboratories) and anti-NR4A1 antibody (Biomatrix Laboratories) were each 1 μg / mL using Samplertech Screener Blotter Mini 56. The concentration was adjusted so that the anti-ATF2 antibody was reacted with the odd lanes from lanes 1 to 12 and 13 to 17 and the anti-NR4A1 antibody was reacted with the even lanes from lanes 1 to 12 and 18 to 22 as the primary antibodies. (1 hour at 37 ° C). After the reaction, an equal volume mixture of HRP-labeled anti-mouse IgG1, IgG2a, IgG2b (both from BETHYL) diluted 15000 times as a secondary antibody was allowed to act for 30 minutes while blocking. For detection, color was developed with Immobilon ^™ western chemiluminescent HRP substrate manufactured by Millipore, and detection was performed using LAS3000 manufactured by Fujifilm.

  As a result of Western blotting, clear detection signals using anti-ATF2 antibody were obtained in lanes 7 (15), 9 (14), 16, and 17 (FIG. 5). On the other hand, no anti-detection signal was obtained in lanes 3 and 5. From these results, it was considered that the antigen recognition site of the anti-ATF2 antibody is in the 29-50 region of the ATF2 partial sequence used.

Table 3 shows the proteins in each lane of FIG.

(Immunoprecipitation of fusion protein of ATF2 partial peptide and target protein-detection by Western blotting)
3 μL of an anti-ATF2 antibody solution adjusted to 100 μg / mL was mixed with 30 μL of protein G sepharose carrier sold by GE Healthcare, and reacted at 4 ° C. for 1 hour with stirring. Thereafter, the carrier was washed with TBS-T by centrifugation and supernatant removal. A protein solution in which NR4A1 was fused downstream of the ATF2 partial peptide 1, 2, 5, 6 or TRX tag described in Example 4 and the concentration was adjusted to 100 μg / mL was added to the carrier thus obtained. The amount of each solution was adjusted to 1 mL using TBS-T, and reacted at 4 ° C. for 1 hour with stirring.
After the reaction, the carrier was washed by centrifugation and supernatant removal, suspended in 30 μL of a sample preparation solution for SDS-PAGE, and heat-treated at 95 ° C. for 10 minutes. After heating, the carrier was precipitated by centrifugation, and SDS-PAGE was performed using the supernatant as a sample (DRC Perfect NT Gel 15-25%, electrophoresis conditions 500 V, 50 mA, 60 min). For Western blotting, the method described in Example 4 was followed. As a result of detection using an anti-ATF2 antibody, the signals derived from the antigens used in lanes 2, 3, 4 and 5 were obtained as shown in FIG. From this, it was confirmed that the epitope site contained in ATF2 partial peptides 1, 2, 5 and 6 was recognized and immunoprecipitation occurred.

Table 4 shows the proteins in each lane of FIG.

It is explanatory drawing which shows insertion of the target protein to the vector which concerns on this invention. (Example 2) which is the figure which showed the SDS-PAGE analysis result which examined the expression of human transcription factor MTA2, NR4A1, NFIB, and PAX8 by the fusion protein expression vector of this invention. It is the figure which showed the SDS-PAGE analysis result which examined the expression of human transcription factor NR4A1 by the expression vector containing various ATF2 partial DNA (Example 3). It is the figure which showed the result of having examined the expression of the various target protein by the expression vector containing ATF2 partial DNA1 and ATF2 partial DNA2 (Example 2 and Example 4). It is the figure which showed the western blotting analysis result which examined the expression of human transcription factor NR4A1 by the expression vector containing various ATF2 partial DNA (Example 5). (Example 6) which is the figure which showed the immunoprecipitation-Western blotting analysis result which examined the expression of human transcription factor NR4A1 by the expression vector containing various ATF2 partial DNA. It is the figure which showed the relationship of ATF2 partial peptide 1-6.

SEQ ID NO: 1 is ATF2 partial DNA 1.
SEQ ID NO: 2 is ATF2 partial DNA2.
SEQ ID NO: 3 is ATF2 partial DNA3.
SEQ ID NO: 4 is ATF2 partial DNA4.
SEQ ID NO: 5 is ATF2 partial DNA5.
SEQ ID NO: 6 is ATF2 partial DNA 6.
SEQ ID NO: 7 is ATF2 partial peptide 1.
SEQ ID NO: 8 is ATF2 partial peptide 2.
SEQ ID NO: 9 is ATF2 partial peptide 3.
SEQ ID NO: 10 is ATF2 partial peptide 4.
SEQ ID NO: 11 is ATF2 partial peptide 5.
SEQ ID NO: 12 is ATF2 partial peptide 6.
SEQ ID NO: 13 is DNA encoding a linker peptide.
SEQ ID NO: 14 is DNA encoding a linker peptide.
SEQ ID NO: 15 is DNA encoding a purification tag.
SEQ ID NO: 16 is DNA encoding a purification tag.
SEQ ID NO: 17 is a primer for PCR.
SEQ ID NO: 18 is a primer for PCR.
SEQ ID NO: 19 is a primer for PCR.
SEQ ID NO: 20 is a primer for PCR.
SEQ ID NO: 21 is a DNA designed for adding an NdeI site to the 5 ′ end of ATF2 partial DNA1.
SEQ ID NO: 22 is DNA designed to add a linker peptide (thrombin digestion site) and a BamHI site to the 3 ′ end of ATF2 partial DNA 1.
SEQ ID NO: 23 is DNA encoding a partial peptide of MTA2 protein.
SEQ ID NO: 24 is DNA encoding a partial peptide of NR4A1 protein.
SEQ ID NO: 25 is DNA encoding a partial peptide of NFIB protein.
SEQ ID NO: 26 is DNA encoding a partial peptide of PAX8 protein.
SEQ ID NO: 27 is DNA encoding a partial peptide of ZNF143 protein.
SEQ ID NO: 28 is DNA encoding a partial peptide of KHSRP protein.
SEQ ID NO: 29 is DNA encoding a partial peptide of EBNA2 protein.
SEQ ID NO: 30 is DNA encoding a partial peptide of STAT3 protein.
SEQ ID NO: 31 is DNA encoding a partial peptide of APEX1 protein.
SEQ ID NO: 32 is DNA encoding a partial peptide of E2F2 protein.
SEQ ID NO: 33 is DNA encoding a partial peptide of HMGA2 protein.
SEQ ID NO: 34 is a DNA encoding a partial peptide of NCOA6 protein.
SEQ ID NO: 35 is DNA encoding a partial peptide of NRIP1 protein.
SEQ ID NO: 36 is DNA encoding a partial peptide of EMG1 protein.
SEQ ID NO: 37 is a histidine tag.
SEQ ID NO: 38 is the full-length amino acid sequence of ATF2 protein.
SEQ ID NO: 39 is a DNA encoding a thrombin digestion site.
SEQ ID NO: 40 is DNA encoding a partial peptide of KIN protein.

Claims (31)

An expression vector for expressing a target protein,
DNA encoding all or part of the transcription factor ATF2 (Activating transcription factor 2) protein,
An insertion site for inserting a DNA encoding the target protein so that the target protein is expressed as a fusion protein with all or part of the ATF2 protein;
An expression vector comprising   The expression vector according to claim 1, wherein the target protein and all or part of the ATF2 protein are expressed as a fusion protein by inserting a DNA encoding the target protein.   The expression vector according to claim 2, further comprising DNA encoding a linker peptide that connects all or part of the ATF2 protein and the target protein in the fusion protein.   The expression vector according to claim 3, wherein the DNA encoding the linker peptide consists of the base sequence set forth in SEQ ID NO: 13, 14, or 39.   Furthermore, the expression vector of any one of Claim 1 to 4 containing DNA which codes the tag protein for a refinement | purification.   The expression vector according to claim 5, wherein the DNA encoding the tag protein for purification consists of the base sequence set forth in SEQ ID NO: 15 or 16.   The expression vector according to any one of claims 1 to 6, wherein the DNA encoding all or part of the ATF2 protein is a DNA comprising the nucleotide sequence according to any of SEQ ID NOs: 1 to 6.   An expression vector deposited as pBMR (deposit number NITE P-522).   Furthermore, the expression vector of any one of Claim 1 to 8 containing DNA which codes the said target protein.   A transformant obtained by introducing the expression vector according to claim 9.   DNA comprising the base sequence set forth in any one of SEQ ID NOs: 1 to 6.   A polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 7 to 12.   An antibody that binds to the polypeptide of claim 12.   Hybridoma cell line deposited under clone number ATF2-00158 (deposit number FERM P-21570).   14. The antibody of claim 13, produced by the cell line of claim 14. The manufacturing method of the target protein including the process of following (a)-(c).
(A) a step of culturing the transformant according to claim 10 in a medium (b) a step of collecting the fusion protein produced in the culture by the step (a) from the culture, and a step (c) ( cleaving the fusion protein collected in b) into all or part of the ATF2 protein and the target protein   The method according to claim 16, wherein in the step (b), the fusion protein is collected by column or immunoprecipitation using an antibody against the ATF2 protein. The manufacturing method of the target protein including the process of following (d)-(f).
(D) a step of culturing the transformant according to claim 10 in a medium (e) a fusion protein produced in the culture by the step (d), comprising all or part of the ATF2 protein, a target protein, And (f) a step of purifying the target protein obtained in step (e).   In the step (f), all or part of the ATF2 protein obtained in the step (e) and a mixture of the target protein and the whole of the ATF2 protein by column or immunoprecipitation using an antibody against the ATF2 protein are used. The method according to claim 18, wherein the target protein is purified by removing a part thereof. A method for producing a fusion protein comprising the following steps (g) to (h):
(G) a step of culturing the transformant according to claim 10 in a medium, and (h) a step of collecting the fusion protein produced in the culture by the step (g) from the culture.   21. The method according to claim 20, wherein in the step (h), the fusion protein is collected by a column using an antibody against the ATF2 protein or immunoprecipitation.   The method according to claim 17, 19 or 21, wherein the antibody is the antibody according to claim 13 or 15.   A method for detecting a fusion protein of all or part of a transcription factor ATF2 protein and a target protein, wherein all or part of the transcription factor ATF2 protein contained in the fusion protein is detected using the antibody according to claim 13 or 15. Recognizing the method.   A method for purifying a fusion protein of all or part of a transcription factor ATF2 protein and a target protein, wherein all or one of the transcription factor ATF2 proteins contained in the fusion protein is obtained using the antibody according to claim 13 or 15. A method for purifying a fusion protein, comprising the step of recognizing a part and purifying the fusion protein.   A DNA encoding a fusion protein containing all or part of the transcription factor ATF2 protein and a target protein.   The DNA according to claim 25, comprising a linker DNA between DNA encoding all or part of the ATF2 protein and DNA encoding the target protein.   The DNA according to claim 26, wherein the linker DNA consists of the base sequence shown in SEQ ID NO: 13, 14, or 39.   The DNA according to any one of claims 25 to 27, comprising DNA encoding a purification tag protein at the carboxy terminus of the DNA encoding the target protein.   The DNA according to claim 28, wherein the DNA encoding the purification tag protein consists of the base sequence shown in SEQ ID NO: 15 or 16.   30. The DNA according to any one of claims 25 to 29, wherein the whole or part of the sequence of the ATF2 protein consists of the base sequence according to any one of SEQ ID NOs: 1 to 6.   A fusion protein comprising all or part of the transcription factor ATF2 protein and the target protein.