KR20130141001A - A novel vector system for isolation and purification of target proteins - Google Patents

Abstract

The present invention relates to a novel vector system for isolating and purifying target proteins, in particular, to a recombinant vector including a part recognizing enterokinase or factor Xa protease, to a cell line and a method for producing recombinant proteins. When introducing genes coding target proteins to the expression vector of the present invention to overexpress, target proteins are obtained in bulk through a simple and economical separating and refining process.

Description

A novel vector system for isolation and purification of target proteins

The present invention relates to a novel vector system for the isolation and purification of a protein of interest, and more particularly to a recombinant vector comprising a site that recognizes enterokinase or factor Xa protease. Cell lines, and methods for producing recombinant proteins.

In order to produce a variety of important proteins that are difficult to obtain in human or animal organs by genetic recombination method in microorganisms, the gene of the target protein is cloned into an expression vector to obtain a recombinant expression vector and transformed into appropriate host cells and cultured. E. coli has the most known information on its genes and metabolic systems compared to other organisms, and is widely used as a host for producing useful foreign target proteins by genetic recombination technology. However, when the target protein is directly expressed in Escherichia coli, it is necessary to make a form in which methionine is added to the N-terminus and convert it to a native protein, which is obtained by adding methionine to the N-terminus of the target protein. When applied to humans or other animals, the immune response (immunogenecity) may be induced or the structure may be unstable to perform the original protein function. In addition, when the expressed protein is highly toxic such as an antibacterial peptide, it may inhibit the growth of the host, and in the case of a polypeptide having a molecular weight of 10,000 or less, it is degraded by protease present in the host cell. As a result, stable mass production becomes difficult.

In order to solve this phenomenon, a gene expression system for fusion expressing an appropriate fusion partner or protein tag at the N-terminus of a target protein has been developed.

The gene expression system obtains a stable transfectant by cloning a foreign protein of interest in an expression vector, transfecting it into animal cells, and selecting colonies resistant to specific antibiotics. It is possible to incubate clones and ultimately increase the expression rate of the target protein.

Another obstacle to high efficiency protein production for proteomics studies is the removal of fusion partners or protein tags. The fusion partner or protein tag is for increased solubility and ease of purification of the protein of interest, but may ultimately be removed in most subsequent studies as it may affect the properties or functions of the protein of interest. Removal of these proteins or peptides is accomplished by site specific proteases. The most important criterion in the selection of such proteases is the high cleavage specificity that cleaves only between the protein of interest and the fusion partner. In the case of degrading enzymes acting within the fusion partner or the target protein, it may cause not only the difficulty of purification but also loss of function or change of properties of the target protein.

Therefore, it is preferable that there is no site of action of the degrading enzyme inside the fusion partner and the target protein, and the protease should have very selective substrate specificity. Thrombin is currently the most widely used protease for the purpose of eliminating fusion partners. However, since it cuts the currently used fusion partner such as NusA into pieces, there is a need for a degrading enzyme having higher specificity of action.

KR 10-1103650 B1, April 15, 2009

The present inventors have made diligent research to find a method for expressing a foreign target protein and efficiently separating it. As a result, the target protein to be expressed is recognized as enterokinase or factor Xa protease. When fused with an amino acid containing a site to express, it was confirmed that the recombinant protein can be efficiently purified and completed the present invention.

It is an object of the present invention to provide an expression vector capable of expressing foreign target proteins and efficiently separating them.

Another object of the present invention is to provide a cell line transformed with the expression vector.

Still another object of the present invention is to provide a method for producing a recombinant protein using the cell line.

In order to achieve the above object, the present invention provides an expression vector capable of expressing and efficiently separating foreign target proteins.

Another object of the present invention to provide a cell line transformed by the expression vector.

Another object of the present invention to provide a method for producing a recombinant protein using the cell line.

Hereinafter, the present invention will be described in detail.

The present invention provides a protein expression cassette of interest comprising nucleotides encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and multiple cloning sites (MCS).

In the present invention, SEQ ID NO: 1 is a site for recognizing Asp-Asp-Asp-Asp-Lys and has a nucleotide sequence described in SEQ ID NO: 3 as an amino acid sequence that recognizes enterokinase (enterokinase), SEQ ID NO: 2 is a site for recognizing Ile-Glu-Gly-Arg and has a nucleotide sequence as set forth in SEQ ID NO: 4 as an amino acid sequence that recognizes factor Xa protease.

In the present invention, multiple cloning sites (MCS) represent synthetic DNA sequences comprising a number of different restriction enzyme sites to allow insertion of DNA encoding the protein of interest.

In the present invention, the target protein expression cassette further includes a nucleotide encoding a protein tag upstream or downstream of the target protein expression cassette in order to increase the purification efficiency of the target protein and express it in an active form having high binding ability with a substrate. can do.

Nucleotides encoding the protein tag (His) ₆ -tag, maltose binding protein, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP16, FLAG, or chloramphenicol acetyl trans It may be selected from the coding sequence of the perase. More preferably, nucleotides encoding (His) ₆ -tags can be used.

More preferably, the target protein expression cassette according to the present invention comprises a nucleotide encoding a (His) ₆ -tag, a nucleotide encoding an amino acid sequence of SEQ ID NO: 1, or a multiple cloning site (MCS). It is included sequentially, and may have one or more nucleotide sequences preferably selected from SEQ ID NOs: 5 to 8.

In the present invention, SEQ ID NO: 5 and SEQ ID NO: 6 is a nucleotide encoding (His) ₆ -tag, the nucleotide of SEQ ID NO: 3 to recognize the enterokinase (enterokinase) and multiple cloning site (multiple cloning site, MCS) DNA fragments having nucleotide sequences complementary to each other as single stranded (single stranded) were prepared and combined to design double stranded DNA fragments.

In addition, the SEQ ID NO: 7 and SEQ ID NO: 8 is a nucleotide encoding (His) ₆ -tag, the nucleotide of SEQ ID NO: 4 to recognize the factor Xa protease and multiple cloning site (multiple cloning site, MCS) ) DNA fragments having single nucleotide sequences complementary to each other as single stranded, were prepared and joined to form a double stranded DNA fragment.

The present invention provides a recombinant vector comprising the target protein expression cassette.

As used herein, a "recombinant vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a gene construct that contains essential regulatory elements operably linked to express the gene insert.

Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. Further, the expression vector includes a selection marker for selecting a host cell containing the vector, and includes a replication origin in the case of a replicable expression vector.

The recombinant vector of the present invention may preferably be prepared by inserting the target protein expression cassette of the present invention into a general E. coli expression vector. In a preferred embodiment of the present invention, pET28a is used as an E. coli expression vector, but is not particularly limited thereto. In general, all E. coli expression vectors that can be used may be used without limitation.

The recombinant expression vectors of the present invention further comprise operably linked promoters and optionally operators capable of directing the transcription and expression of genes downstream of E. coli. The expression may be constitutive or controllable, but is preferably controllable, and may be induced at high efficiency by, for example, addition of an expression inducer such as IPTG (isopropyl-β-thiogalactoside) to overexpress the target protein. It is desirable to be able to.

In a preferred embodiment of the present invention, a pET28a vector, which is an E. coli expression vector, uses a nucleotide encoding the (His) ₆ -tag, a nucleotide encoding an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and a multiple cloning site (multiple) Recombinant vectors were prepared by inserting the target protein expression cassette DNA fragments to which cloning sites (MCS) were sequentially connected, and named "pEk", or "pFxa".

In the present invention, the recombinant vector preferably has a cleavage map of FIG. 2, but is not limited thereto.

The recombinant vector according to the present invention may further include a gene encoding a protein of interest, and by expressing such a recombinant vector it is possible to express the protein of interest with high efficiency.

The target protein generally includes a physiologically active polypeptide, and is a hormone, a cytokine, an interleukin, an interleukin binding protein, an enzyme, an antibody, a growth factor, a transcriptional regulator, a blood factor, a vaccine, a structural protein, a ligand protein, or Various proteins such as receptors, cell surface antigens, receptor antagonists and derivatives and analogs thereof can be exemplified, specifically, human growth hormone, interferon and interferon receptors, colony stimulating factors, interleukins, erythropoietin, insulin, Angiotensin, bone forming growth factor, B cell factor, T cell factor, nerve growth factor, cell surface antigen, monoclonal antibody and virus derived vaccine antigen and the like.

The recombinant vector is inserted into a host cell to form a transformant. Since expression levels and expressions of proteins vary depending on the host cell, a host cell may be selected and used most suitable for the purpose.

Examples of host cells include Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus but are not limited to, prokaryotic host cells such as mirabilis or Staphylococcus . In addition, fungi (eg Aspergillus ), yeast (eg Pichia pastoris , Saccharomyces Saccharomyces) cerevisiae), investigating car break in Rome Seth (Schizosaccharomyces), Castello La Neuro Chrysler Corporation (Neurospora can be used lower eukaryotic cells, insect cells, plant cells, mammalian cells, such as in higher eukaryotic origin, including such as crassa)) as a host cell.

Transformation into a host cell in the present invention includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and can be carried out by selecting a suitable standard technique according to the host cell as is known in the art. These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and dry / inhibited mediated transformation methods and the like.

The present invention provides a protein production method for culturing a transformant expressing a recombinant vector in a nutrient medium.

More specifically, (1) inserting a gene encoding a target protein into the multiple cloning site of the pEk vector, or pFxa vector plasmid; (2) transforming the plasmid into E. coli and culturing in a nutrient medium; And (3) isolating and purifying the fusion protein from the culture solution; It includes, provides a method for producing a target protein.

The medium and the culture conditions may be appropriately selected depending on the host cell. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and the mass production of proteins during the culture.

The production method may further comprise the step of purifying the protein, it is possible to isolate and purify by conventional biochemical separation techniques. These include electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various variations and complex methods thereof. This is available but not limited to.

The present invention can produce a fusion protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and the target protein according to the present invention by culturing the transformant expressing the recombinant vector in a nutrient medium.

The fusion protein (fusion protien) refers to a protein in which another protein is linked or the other amino acid sequence is added to the N-terminus or C-terminus of the sequence of the original target protein.

The target protein generally includes a physiologically active polypeptide, and is a hormone, a cytokine, an interleukin, an interleukin binding protein, an enzyme, an antibody, a growth factor, a transcriptional regulator, a blood factor, a vaccine, a structural protein, a ligand protein, or Various proteins such as receptors, cell surface antigens, receptor antagonists and derivatives and analogs thereof can be exemplified, specifically, human growth hormone, interferon and interferon receptors, colony stimulating factors, interleukins, erythropoietin, insulin, Angiotensin, bone forming growth factor, B cell factor, T cell factor, nerve growth factor, cell surface antigen, monoclonal antibody and virus derived vaccine antigen and the like.

In the present invention, SEQ ID NO: 1 is a site for recognizing Asp-Asp-Asp-Asp-Lys, characterized in that the amino acid sequence of five amino acids that recognize enterokinase (enterokinase), SEQ ID NO: 2 Ile-Glu It is a site for recognizing factor Xa protease as a site for -Gly-Arg recognition.

The fusion protein according to the present invention may further include a protein tag upstream or downstream of the fusion protein in order to increase the purification efficiency of the fusion protein and express it in an active form having high binding strength with a substrate.

The protein tag is selected from (His) ₆ -tag, maltose binding protein, hemagglutinin A, beta-galactosidase, thymidine kinase, transferrin, myc-tag, VP16, FLAG, or chloramphenicol acetyl transferase Can be. More preferably, (His) ₆ -tag can be used.

The present invention provides a polynucleotide encoding a fusion protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and the target protein.

In the present invention, the polynucleotide encoding the fusion protein includes a nucleic acid molecule encoding an amino acid of SEQ ID NO: 1 or SEQ ID NO: 2 and a nucleic acid molecule encoding a target protein, the polynucleotide encoding the fusion protein It may further comprise nucleotides encoding a protein tag upstream or downstream.

More preferably, the polynucleotide encoding the fusion protein comprises a nucleotide set forth in SEQ ID NO: 3 or SEQ ID NO: 4 and a nucleic acid molecule encoding a target protein, and (His) ₆ − upstream of the polynucleotide encoding the fusion protein. Nucleotides that encode a tag.

In one embodiment of the present invention, the fusion proteins pEk_avp and pFxa_avp were prepared by fusing AVP protein which is the target protein to recombinant vectors "pEk vector" and "pFxa vector". The monoclonal cell line transformed with the fusion protein was obtained and mass cultured to analyze the total protein of Escherichia coli culture by SDS-PAGE. As a result, it was confirmed that about 18 kDa protein was efficiently expressed in E. coli by IPTG induction. It was.

Recombinant expression vector according to the present invention has the advantage that can block in advance the loss of function or characteristic change of the protein due to the residual amino acid because there is no residual amino acid when cleaving the protein tag and the target protein.

Introducing and overexpressing a gene encoding a target protein in the recombinant expression vector according to the present invention, there is an advantage that a large amount of the target protein can be obtained through a simple and economical separation and purification process.

Figure 1 shows the binding of the pET28a vector with a site that recognizes enterokinase (enterokinase) or factor Xa protease (factor Xa protease),
2 shows a cleavage map of the recombinant vector according to the present invention,
(A: pEk vector, B: pFxa vector)
Figure 3 shows the nucleotide sequence of the fusion protein (pEk_avp) prepared by fusion of the target protein AVP protein to the recombinant vector pEk vector according to the present invention,
Figure 4 shows the nucleotide sequence of the fusion protein (pFxa_avp) prepared by fusion of the target protein AVP protein to the recombinant vector pFxa vector according to the present invention,
5 is a result confirming the expression of the protein in E. coli of the recombinant vector according to the present invention.
(1: pFxa_avp, 2: pEk_avp, 3: pET3a_avp)

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and it is apparent to those skilled in the art that various changes or modifications can be made within the idea and scope of the present invention.

Here, unless otherwise defined in the technical terms and scientific terms used, those having ordinary skill in the art to which the present invention belongs have a generally understood meaning.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

Example 1 Preparation of DNA Fragment

In order to make a cassette structure for the expression of the protein of interest, the nucleotide of SEQ ID NO: 3 from 5 amino acids Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 1) having an recognition site for enterokinase (GAT GAC GAT GAC) Nucleotides of SEQ ID NO: 4 (ATA GAG GGT CGT) were synthesized from four amino acids Ile-Glu-Gly-Arg (SEQ ID NO: 2) having a recognition site for AAG) and factor Xa protease.

(His) ₆ -tag (CAT CAT CAT CAC CAC) was prepared at the N-amino acid end of the fusion partner to enable protein purification using affinity chromatography. And a nucleotide of SEQ ID NO: 3 from 5 amino acids Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 1) having a recognition site for enterokinase to remove (His) 6-tag to obtain pure protein (GAT GAC GAT GAC AAG) and factor Xa protease to produce four amino acids Ile-Glu-Gly-Arg (SEQ ID NO: 2) having a recognition site for the fusion of various target proteins Easily used multiple cloning sites (MCS) were made. Nco 1 (5` ends) and Xho 1 (3` ends), which are base sequences of restriction enzymes, were added to the ends of each primer.

The DNA fragment was cloned using the primers of Table 1 below.

The primer of SEQ ID NO: 5, the primer of SEQ ID NO: 6, the primer of SEQ ID NO: 7, and the primer of SEQ ID NO: 8 in single-stranded state dissolved in purified distilled water were added to a new tube in equal amounts to each other It was left for 10 minutes at 55 ℃ to bind to thereby secure a double-stranded DNA fragments.

The obtained double-stranded DNA fragment has restriction enzyme sites of Nco 1 and Xho 1 at each end, so the restriction enzymes Nco 1 and Xho are removed to remove the vector-specific protein tag and multiple cloning sites in the pET28a (Novagen, USA) vector. After treating with 1, the primer prepared for the expression of the target protein was finally inserted.

Example 2 Preparation of Expression Vector

To prepare a pET28a cloning vector comprising a double-stranded DNA fragment prepared in Example 1, pET28a (Novagen, USA) cut with restriction enzymes Nco1 and Xho1 and double-stranded form prepared in Example 1 DNA fragment was added to the T4 DNA ligase (T4 DNA ligase, Roche Co.) and buffer (650 mM Tris-HCl, pH 8.3; 50 mM MgCl ₂ ; 10 mM KCl; 1% Triton X-100) After mixing, the mixture was reacted at 16 ° C. for 16 to 24 hours to prepare a recombinant plasmid vector having a DNA fragment inserted into the pET28a plasmid DNA vector, which was termed a “pEk vector” or a “pFxa vector”. See FIG.

Example 3 Confirmation of Protein Expression in Escherichia Coli

The fusion proteins pEk_avp (SEQ ID NO: 9) and pFxa_avp (SEQ ID NO: 10) were fused to the recombinant vectors "pEk vector" and "pFxa vector" prepared in Example 2 to the AVP protein of interest.

The AVP protein was recombined from the RC216816 plasmid (Origen, USA) with the rest of the active site (30-165 amino acids) except for the signal peptide in the human Arginine vasopressin (AVP) protein.

Primers were prepared to use Nde 1 and Bam H1 among the multiple cloning sites of the recombinant vectors “pEk vector” and “pFxa vector”. The primers prepared a forward primer (GCG CAT ATG TAC TTC CAG AAC TGC CCG AGG) containing a base sequence of Nde 1 and a reverse primer (GCG GGA TCC TCA GTA GGC GTC GGG CTG GG) containing a base sequence of Bam H1. Each primer and RC216816 plasmid were used to amplify the AVP protein by polymerase chain reaction under conditions of 40 seconds at 95 ° C, 40 seconds at 55 ° C, and 72 minutes at 72 ° C.

The pEk vector and pFxa vector and the AVP polymerase chain reaction product were treated with Nde 1 and Bam H1 restriction enzymes, and the DNA fragments of the restriction enzyme-treated AVP, pEk vector and pFxa vector were T4 DNA ligase (T4). DNA ligase, Roche Co.) and buffer (650 mM Tris-HCl, pH 8.3; 50 mM MgCl ₂ ; 10 mM KCl; 1% Triton X-100) were added and mixed, followed by 16 to 24 hours at 16 ° C. During the reaction, pEk_avp and pFxa_avp recombinant fusion proteins in which AVP fragments were inserted in the pEk vector and pFxa vector plasmid DNA vectors were prepared.

In order to compare the expression level of the protein with the recombinant expression vector of Example, pET3a vector, which is a conventionally used expression vector, was used, and the pET3a_avp recombinant fusion protein was prepared by inserting the AVP fragment in the same manner as described above.

Each of the prepared fusion proteins was transformed into Escherichia coli, and then inoculated in LB-Broth media, followed by shaking culture. When the concentration of the cultured cells showed an absorbance of 0.6 at 600 nm, the final 1 mM IPTG was added to the culture medium. After induction, shaking culture was continued at 30 ° C. for a certain time.

As a result of analyzing the total protein of the cultured E. coli culture by SDS-PAGE, when a gene encoding the target protein was introduced into the recombinant expression vector according to the embodiment by induction of IPTG as shown in FIG. It was confirmed that the target protein of about 18 kDa can be obtained in large quantities through a simple and economical separation and purification process compared to the existing expression vector.

<110> KOREA RESEARCH INSTITUTE OF STANDARDS AND SCIENCE <120> A Novel vector system for isolation and purification of target          proteins <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 5 <212> PRT <213> enterokinase <400> 1 Asp Asp Asp Asp Lys   1 5 <210> 2 <211> 4 <212> PRT Factor Xa <400> 2 Ile Glu Gly Arg   One <210> 3 <211> 15 <212> DNA <213> enterokinase <400> 3 gatgacgatg acaag 15 <210> 4 <211> 12 <212> DNA Factor Xa <400> 4 atagagggtc gt 12 <210> 5 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> 6His-Enterokinase primer <400> 5 catgggccat catcatcatc accacgatga cgatgacaag catatgaagc ttggtaccga 60 gctcgaattc ggatcctcta gac 83 <210> 6 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> 6His-Enterokinase primer <400> 6 tcgagtctag aggatccgaa ttcgagctcg gtaccaagct tcatatgctt gtcatggtca 60 tcgtggtgat gatgatgatg gcc 83 <210> 7 <211> 80 <212> DNA <213> Artificial Sequence <220> 6His-Factor Xa primer <400> 7 catgggccat catcatcatc accacataga gggtcgtcat atgaagcttg gtaccgagct 60 cgaattcgga tcctctagac 80 <210> 8 <211> 80 <212> DNA <213> Artificial Sequence <220> 6His-Factor Xa primer <400> 8 tcgagtctag aggatccgaa ttcgagctcg gtaccaagct tcatatgacg accctctatg 60 tggtgatgat gatgatggcc 80 <210> 9 <211> 480 <212> DNA <213> pEk_avp fusion protein <400> 9 catcatcatc atcaccacga tgacgatgac aagcatatgt acttccagaa ctgcccgagg 60 ggcggcaaga gggccatgtc cgacctggag ctgagacagt gcctcccctg cggccccggg 120 ggcaaaggcc gctgcttcgg gcccagcatc tgctgcgcgg acgagctggg ctgcttcgtg 180 ggcacggctg aggcgctgcg ctgccaggag gagaactacc tgccgtcgcc ctgccagtcc 240 ggccagaagg cgtgcgggag cgggggccgc tgcgccgcct tcggcgtttg ctgcaacgac 300 gagagctgcg tgaccgagcc cgagtgccgc gagggctttc accgccgcgc ccgcgccagc 360 gaccggagca acgccacgca gctggacggg ccggccgggg ccttgctgct gcggctggtg 420 cagctggccg gggcgcccga gcccttcgag cccgcccagc ccgacgccta ctgaggatcc 480                                                                          480 <210> 10 <211> 477 <212> DNA <213> pFxa_avp fusion protein <400> 10 catcatcatc atcaccacat agagggtcgt catatgtact tccagaactg cccgaggggc 60 ggcaagaggg ccatgtccga cctggagctg agacagtgcc tcccctgcgg ccccgggggc 120 aaaggccgct gcttcgggcc cagcatctgc tgcgcggacg agctgggctg cttcgtgggc 180 acggctgagg cgctgcgctg ccaggaggag aactacctgc cgtcgccctg ccagtccggc 240 cagaaggcgt gcgggagcgg gggccgctgc gccgccttcg gcgtttgctg caacgacgag 300 agctgcgtga ccgagcccga gtgccgcgag ggctttcacc gccgcgcccg cgccagcgac 360 cggagcaacg ccacgcagct ggacgggccg gccggggcct tgctgctgcg gctggtgcag 420 ctggccgggg cgcccgagcc cttcgagccc gcccagcccg acgcctactg aggatcc 477

Claims (14)

A target protein expression cassette, comprising a multiple cloning site (MCS) and a nucleotide encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 3. The method of claim 2,
The SEQ ID NO: 1 or SEQ ID NO: 2 is the target protein expression cassette, characterized in that the site that recognizes enterokinase (enterokinase) or factor Xa protease (factor Xa protease). The method of claim 1,
The cassette further comprises a nucleotide encoding (His) ₆ -tag upstream or downstream of the cassette. The method of claim 3, wherein
The cassette of interest protein expression cassette, characterized in that it has one or more nucleotide sequence selected from SEQ ID NO: 5 to 8. A recombinant vector comprising the desired protein expression cassette of any one of claims 1 to 4. 6. The method of claim 5,
The vector is a recombinant vector, characterized in that the gene encoding the target protein is additionally operably linked to the target protein expression induction cassette. 6. The method of claim 5,
The vector has a cleavage map of FIG. 2. A cell line transformed with a recombinant vector according to any one of claims 5 to 7. A method of producing a protein comprising culturing the cell line of claim 8 and expressing the protein. The method of claim 9,
The production method of the protein further comprises the step of purifying the protein. A fusion protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and the target protein produced by the protein production method according to claim 9. 12. The method of claim 11,
Wherein SEQ ID NO: 1 or SEQ ID NO: 2 is a fusion protein, characterized in that the site that recognizes enterokinase (enterokinase) or factor Xa protease (factor Xa protease). 12. The method of claim 11,
The fusion protein further comprises a (His) ₆ -tag upstream or downstream of the fusion protein. A polynucleotide encoding a fusion protein according to any one of claims 11 to 13.

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