KR20160088259A - Method for Preparing Active Arginine Deiminase - Google Patents

Abstract

The present invention relates to a method for producing an active arginine deiminase (ADI) by using malate dehydrogenase (MDH) or an elongation factor Ts (Tsf) as a fusion partner and, more specifically, to a method for producing an active ADI having improved water-solubility and folding properties by using a gene construct or an expression vector including an MDH or Tsf gene as a fusion partner. A method for producing an ADI by using MDH or Tsf as a fusion partner improves water-solubility and expression of an ADI which is difficult to be expressed, so an original anticancer enzyme effect can be maintained. Accordingly, the method is useful in developing and producing an anticancer treating agent.

Description

Method for preparing active arginine deiminase {Method for Preparing Active Arginine Deiminase}

The present invention relates to a method for preparing active arginine deiminase (ADI) using Mdh (Malate dehydrogenase) or Tsf (Elongation factor Ts) as a fusion partner, and more particularly, to a fusion partner of Mdh or Tsf genes. It relates to a method for preparing an active arginine deiminease (ADI) with improved water solubility and folding using an expression vector or a gene construct including as.

Proteins produced by biotechnology can be broadly classified into pharmaceutical and research proteins such as immunomodulatory and enzyme inhibitors and hormones, and industrial proteins such as diagnostic proteins or reaction additive enzymes. Development and industrialization are being promoted.

Arginine deiminase (ADI) is a bacterial enzyme that breaks down arginine, an amino acid essential for cancer cell metabolism, to produce citrulline and ammonia. Arginine deimase is a hepatocellular carcinoma, melanoma, and leukemia due to the effect of inhibiting cancer apoptosis and cancer angiogenesis and neutralizing endotoxin through inhibition of nitric oxide synthesis. It is known to have anti-cancer properties against leukemia and prostate cancer. According to previous research results, it is known that deprivation of arginine from cancer cells with arginine deimase causes mitochondrial dysfunction, reactive oxygen species, nuclear DNA leakage, and chromatin autophagy to kill cancer cells (Chun A). Changou et al., PNAS, 111(39) 14147-14152). Due to its anticancer activities such as inhibition of cancer proliferation and generation of cancer angiogenesis, arginine deiminease is attracting attention as an effective enzyme in treating cancer. Actually, ADI-PEG20 drug was made by attaching a polyethylene glycol chain with an average molecular weight of 20 kDa to arginine deiminase at Polaris Pharmaceuticals in San Diego, USA, and through this, liver cancer (clinical phase 3), It is applied in clinical trials for the treatment of various cancers such as melanoma (phase 2 clinical trial), prostate cancer, lung cancer, prostate cancer, and oral cancer (hereinafter, phase 1 clinical trial). Therefore, genetic information is well known and various vector systems can be applied, and using microorganisms such as Escherichia coli, which has the advantage of being able to rapidly cultivate in a relatively inexpensive medium at high concentration, is used for medical and industrial purposes such as arginine deiminase (ADI). As a result, it is necessary to construct a system capable of producing a useful recombinant protein.

In the production of recombinant proteins in E. coli, various expression vectors with strong inducible promoters have been developed and used for the production of foreign proteins. However, when Escherichia coli is used as a host cell, the protein to be produced is often degraded by proteolytic enzymes in Escherichia coli, resulting in a low yield. In particular, this tendency is known to be severe in the expression of small-sized polypeptides with a molecular weight of 10 kDa or less have. In addition, in general, E. coli forms an insoluble aggregate (inclusion body) when overexpressing a recombinant protein because protein transcription and translation occur almost simultaneously, and in the case of a polypeptide expressed as an aggregate, it is folded. (folding) Intermolecular disulfide bond (intermolecular disulfide bond) or hydrophobic interaction (hydrophobic interaction) to other protein impurities of the host cell (chaperon (chaperon), ribosome (ribosome), initial factor, etc.) Due to non-selective binding, there is a disadvantage that the purity in the aggregate of the target polypeptide is lowered. In addition, in order to make the expressed protein into an active form, it must be dissolved with a denatured substance such as guanidine-hydrochloride or urea, and then diluted with a refolding process. There is a problem that the production yield decreases, such as not folding (Marston FA et al., Biochem J 240(1):1-12, 1986).

A low-temperature expression method that increases the acceptability of the target protein by slowing the protein expression rate of E. coli in order to obtain a high yield of the active recombinant protein in E. coli (Hammarstrom et al., Protein Sci. 11:313-321, 2002), Using an advanced form of promoter capable of enhancing the affinity of mRNA or optimizing induction conditions (Qing et al., Nat Biotechnol. 22:877-882, 2004) and molecular chaperones or protein folding regulators A method of simultaneously expressing a target protein (de Marco & De Marco, J Biotechnol. 109:45-52, 2004) has been attempted, but the method of expressing by fusing a fusion partner to the amino terminus of the target protein is most commonly used. have. When the fusion partner is fused and expressed, it has the advantage of inducing the target protein to be water-soluble, and by removing the fusion partner using an enzyme such as enterokinase, the amino-terminal methionine problem can be solved.

In fact, several fusion partners that induce water-soluble expression of foreign proteins in E. coli have been studied and reported over the past several years (Esposito & Chatterjee, Curr Opin Biotechnol. 17:353-358 2006; Kapust & Waugh, Protein Sci. 8: 1668-1674, 1999; Sachdev & Chirgwin, Biochem. Biophys. Res. Commun. 244:933-937, 1998). Representative fusion partners that have been studied the most include maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (Trx), and NusA. . In the case of maltose-binding protein, a wide hydrophobic gap made by gathering hydrophobic amino acids in the part involved in dimer formation effectively hides the hydrophobic part of the newly synthesized protein, preventing the target protein from becoming insoluble aggregates. It is known that singleness helps disulfide binding of the target protein, and in the case of NusA, when it is expressed in excess in E. coli, the ability to fold into an active form is very good, so it is known that it induces the correct folding of the target protein that is subsequently expressed (Bach H et al. , J Mol Biol 312:79-93, 2001; Edward RL et al., Nat Biotechnol 11:187-193, 1993; Davis GD et al., Biotechnol Bioen g 65:382-388, 1999).

Such a fusion partner has the advantage of being able to easily purify a target protein expressed by fusion using an affinity chromatography method, and is known to help folding of a target protein using different molecular biological properties. However, since these fusion partners are relatively larger than the target protein, the yield of the target protein is significantly lowered depending on the size of the fusion portion, and there are disadvantages that they do not work universally for both medical or industrially useful proteins. . In addition, there are cases where the target protein is also produced in the form of a dimer by forming a dimer, and even if the target protein is induce water-soluble expression, it fails to induce the expression into an active form that performs its own function. In many cases, there is a problem in that there is a process irrationality in which the removal process of the fusion partner must be added in order to be used for a suitable purpose. In addition, proteins that are very important commercially and medically are secretory proteins and membrane proteins, which are difficult-to-express proteins that make insoluble aggregates when expressed in Escherichia coli. have. In order to overcome this, it is important to develop an expression system to increase the expression efficiency of an unexpressed protein by utilizing the E. coli system, which has various advantages, and to secure the original base technology for this.

Accordingly, the present inventors have made diligent efforts to construct an active expression system of a non-expressing bacterial enzyme having anticancer efficacy, and as a result of fusion expression with arginine deiminase (ADI) using Mdh or Tsf as a fusion partner, It was confirmed that the water-soluble and active expression of the arginine deimase enzyme was remarkably improved, and the present invention was completed.

An object of the present invention is to provide an expression vector or gene construct for the production of a recombinant protein capable of improving the solubility and folding of arginine deiminase (ADI) by using the Mdh or Tsf gene as a fusion partner. There is it.

Another object of the present invention is to provide a recombinant microorganism into which the expression vector or gene construct is introduced, and a method for producing a recombinant protein using the same.

Another object of the present invention is to provide a recombinant protein in which Mdh or Tsf prepared by the above method and arginine deiminase (ADI) are fused.

In order to achieve the above object, the present invention provides a gene construct in which the gene encoding the ADI protein and the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 are linked.

The present invention also provides an expression vector for production of recombinant ADI comprising the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 as an ADI gene and a fusion partner.

The present invention also provides a recombinant microorganism into which the gene construct or the expression vector has been introduced.

The present invention also provides a method for producing a recombinant protein comprising the step of culturing the recombinant microorganism to induce expression of the recombinant ADI protein, and then recovering the recombinant ADI protein.

The present invention also provides a recombinant protein prepared by the above method and in which Mdh or Tsf and ADI are fused.

The manufacturing method of arginine deiminease (ADI) using Mdh or Tsf according to the present invention as a fusion partner can improve the water solubility and expression rate of poorly expressed ADI to maintain the natural anticancer enzyme activity, and develop and produce anticancer therapeutic agents using the same. Useful for

1 is a schematic diagram of a fusion expression vector (B) in which a polynucleotide encoding an enterokinase recognition site is linked between an expression vector containing only an ADI gene (A) and a fusion partner (Mdh, Tsf, and GST) gene and an ADI gene. to be.
Figure 2 shows the results of ADI expression alone and fusion partners (Mdh, Tsf and GST) and ADI fusion expression by SDS-PAGE.
3 is a result of comparing the expression level and water-soluble expression level of ADI expression alone and fusion partners (Mdh, Tsf and GST) and ADI fusion expression.
Figure 4 is a result of SDS-PAGE analysis of active ADI from which fusion partners (Mdh and Tsf) have been removed.
5 is a result showing the activity of the active ADI from which the fusion partners (Mdh and Tsf) have been removed.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In E. coli, the protein of interest is often expressed as an insoluble aggregate. In other words, transcription and transfer occur almost simultaneously in the E. coli expression system, and when the recombinant protein is overexpressed, the adjacent hydrophobic (hydrophobic) is caused by non-specific binding between the target protein or the folding intermediates of other proteins in the E. coli host cell. ) Polypeptide chains are known to form insoluble aggregates known as inclusion bodies. The activity is maintained even under conditions that inhibit the folding of the protein, and the protein with excellent expression level can be involved in the biomechanism of E. coli to overcome the misfolding of the protein, and because the protein structure is stable and has excellent self-folding ability, it is fused. Can be used effectively as a partner. Among the E. coli fusion and expression partners, the fusion expression partner known to have the highest efficiency is Maltose binding protein ('MBP'; Bedouelle and Duplay, Eur J Biochem , 1998). However, MBP, which is known to be effective against a large number of proteins, does not show an effect of improving water-soluble expression against many proteins (Hammarstrom et al., J Struct Funct Genomics, 2006).

In the present invention, by using a malate dehydrogenase (Mdh; Malate dehydrogenase) or Tsf (Enlongation factor Ts) gene as a fusion partner to produce an active form of arginine deiminase (ADI), insoluble aggregates can be converted to denaturants or activators. A method for producing a high value-added medical protein capable of maintaining the original activity and structure of an enzyme without using it has been provided.

Accordingly, in one aspect, the present invention relates to a gene construct in which the gene encoding the ADI protein and the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 are linked.

In the present invention, a polynucleotide encoding a protein cleavage enzyme recognition site may be linked between the ADI gene and the Mdh gene or the Tsf gene.

In addition, the ADI gene and the Mdh gene or the Tsf gene may be operably linked with a polynucleotide encoding a tag for isolation and purification.

In another aspect, the present invention relates to an expression vector for the production of recombinant ADI comprising the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 as an ADI gene and a fusion partner.

In the present invention, a polynucleotide encoding a protein cleavage enzyme recognition site may be linked between the ADI gene and the Mdh gene or the Tsf gene as a fusion partner.

In addition, the ADI gene and the Mdh gene or the Tsf gene may be operably linked with a polynucleotide encoding a tag for separation and purification.

In the present invention, the ADI gene may be characterized in that it is represented by SEQ ID NO: 13.

The expression vector of the present invention may express the fusion partner Mdh or Tsf and ADI as a recombinant protein by including the fusion partner Mdh or Tsf gene and the target protein ADI gene in a series of sequence.

As used herein, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are currently the most commonly used form of vectors, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferred to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a replication starting point that enables efficient replication to contain several to hundreds of plasmid vectors per host cell, and (b) host cells transformed with plasmid vectors are selected. It has a structure that includes an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method. After ligation, the vector must be transformed into an appropriate host cell. Transformation can be easily achieved using the calcium chloride method or electroporation (Neumann, et al., EMBO J., 1:841, 1982) or the like.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used. The skeleton vector that can be used in the method of the present invention is not particularly limited thereto, but various vectors capable of transforming E. coli selected from the group consisting of pT7, pET/Rb, pGEX, pET28a, pET-22b(+) and pGEX are used. Can be used.

A base sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. This may be a gene and regulatory sequence(s) linked in a manner that allows gene expression when an appropriate molecule (eg, a transcriptional activating protein) is bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide, and a promoter or enhancer is a sequence If it affects the transcription of the coding sequence, or if the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence, or if the ribosome binding site is arranged to facilitate translation coding Operably linked to the sequence. In general, "operably linked" means that the DNA sequence to which it is linked is in contact, and in the case of a secretory leader, is contacted and is in the reading frame. However, the enhancer does not need to be in contact. The ligation of these sequences is carried out by ligation (linkage) at a convenient restriction enzyme site. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.

As is well known in the art, in order to increase the level of expression of a transgene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that exert a function in the selected expression host. Preferably, the expression control sequence and the corresponding gene are included in a single recombinant vector including a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector must further contain an expression marker useful in the eukaryotic expression host.

Host cells transformed with the above-described recombinant vector constitute another aspect of the present invention. As used herein, the term "transformation" means that DNA is introduced into a host so that the DNA becomes replicable either as an extrachromosomal factor or by chromosomal integrity.

Of course, it should be understood that not all vectors function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art can make an appropriate selection among various vectors, expression control sequences, and hosts without departing from the scope of the present invention without undue experimental burden. For example, when choosing a vector, you must consider the host, because the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.

Therefore, in another aspect, the present invention is a gene construct in which the gene encoding the ADI protein and the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 are linked or the ADI gene and It relates to a recombinant microorganism into which an expression vector for the production of recombinant ADI including the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 is introduced as a fusion partner.

In the present invention, the gene construct may be inserted into a chromosome of a host cell.

It will be apparent to those skilled in the art to which the present invention pertains that even when the gene is inserted into the genomic chromosome of a host cell, it will have the same effect as when the recombinant vector is introduced into a host cell as described above.

In the present invention, as a method of inserting the gene onto the chromosome of a host cell, a conventionally known gene manipulation method can be used, for example, a retroviral vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector , A method of using a poxvirus vector, a lentiviral vector, or a non-viral vector.

In another aspect, the present invention is a gene construct in which the gene encoding the ADI protein and the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 are linked, or the ADI gene and the fusion partner Induce expression of the recombinant protein by culturing a recombinant microorganism into which the expression vector for producing recombinant ADI including the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 has been introduced, and then recovering it. It relates to a method for producing a recombinant ADI protein comprising.

In the present invention, it may be characterized in that it further comprises the step of removing Mdh or Tsf from the recombinant protein.

When the target protein to be fused with the fusion partner is an industrial protein, the fusion partner may reduce the function of the target protein, and in the case of a medical protein, it is preferable to remove the fusion partner because it may cause an antigen-antibody reaction. At this time, the protein cleavage enzyme recognition site may be used alone or by sequentially connecting any two or more of the Xa factor recognition site, enterokinase recognition site, Genenase I recognition site, or Furin recognition site. have.

In addition, in order to facilitate separation and purification of the recombinant protein, a polynucleotide encoding a tag for separation and purification may be operably linked to the fusion partner of the vector of the present invention or the gene of the target protein. At this time, the tag for separation and purification may be used alone, such as GST, poly-Arg, FLAG, poly-His and c-myc, or may be used by connecting any two or more in sequence. In one embodiment of the invention, the vector of the present invention comprises a tag for separation and purification in a pT7 skeleton vector, a gene of the fusion partner, and a gene of the target protein in a series of order, thereby providing the tag for separation and purification, the fusion partner and the protein of interest. It is possible to express the containing recombinant protein.

The gene of the target protein can be cloned through a restriction enzyme site, and when a polynucleotide encoding a protein cleavage enzyme recognition site is used, it is linked in frame with the polynucleotide, and after secretion of the target protein By cutting with a protein cleavage enzyme, it can be made to produce the desired protein in its original form.

As used herein, the term "target protein" or "heterologous protein" is a protein that a manufacturer wants to produce in large quantities. In a transformant, a polynucleotide encoding the protein is inserted into a recombinant expression vector. It means any protein that can be expressed.

In addition, in the present invention, "recombinant fusion protein" refers to a protein in which the target protein ADI and Mdh or Tsf are fused.

In another aspect, the present invention relates to a recombinant protein prepared by the above method and fused with Mdh or Tsf and ADI.

The method of producing an ADI protein using Mdh or Tsf of the present invention as a fusion partner can maintain the unique tertiary structure and intrinsic enzymatic activity of the fusion-expressed ADI, so it can be widely used in the production of high value-added protein drugs. There is this.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Analysis of changes in E. coli protein body due to environmental stress

Conditions that fusion partners must have include high expression rates and effective three-dimensional structure formation. In order to form a particularly effective three-dimensional structure, structural stability of the protein must be premised. Accordingly, a change in the expression environment of the production strain was induced, and the expression level and the protein in which the function was maintained were analyzed.

1-1: Preparation of water-soluble protein for proteomic analysis

For proteomic analysis, the E. coli BL21 (DE3) strain was selected, and a portion of the seed culture medium cultured overnight was inoculated into LB medium, followed by incubation at 37° C. and 130 rpm. When the OD ₆₀₀ of the culture medium reaches 0.5, guanidine hydrochloride (guanidine hydrochloride, hereinafter'GdnHCl') and 2-hydroxyethyl disulfide (hereinafter '2HEDS'), which are substances that inhibit the three-dimensional folding of the protein, were added to a final concentration of 0.1 mM and After the addition of 10 mM to the E. coli culture medium, the culture was further cultured for 3 hours, and the comparative E. coli was cultured for the same time without the addition of GdnHCl and 2HEDS. The cultured E. coli BL21 (DE3) culture was centrifuged at 6000 rpm and 4° C. to recover the bacterial precipitate and washed twice with pH 8.0 and 40 mM Tris buffer. 500 µl of the washed E. coli sediment was disrupted (lysis buffer; 8 M urea, 4% (w/v) CHAPS, 40 mM Tris, protease inhibitor cocktail) ), and then crushed using an ultrasonic crusher. After crushing the cells, centrifugation was performed at 12,000 rpm for 60 minutes at 4°C to remove the crushed cells, and then the supernatant was separated.

1-2: 2-dimensional gel electrophoresis for proteomic analysis

The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit. The supernatant containing 30 mg of water-soluble protein was fractionated and rehydration solution (2 M thiourea), 8 M urea (urea), 4% w/v Chaps (CHAPS), 1% w/ v DTT, 1% w/v mobile positive electrolyte (carrier ampholyte), pH 4.7). The one-dimensional isoelectric point separation process was performed using a Bio-Rad Protein IEF cell electrophoresis system, and an immobilized pH gradient (IPG) gel strip (17 cm, ReadyStrip) in the range of linear pH 4-7 was used. The protein contained in the rehydration solution was rehydrated overnight. 500 V for 2 hours, 1000 V for 30 minutes, 2000 V for 30 minutes, 4000 V for 30 minutes, and 8000 V for 70000 VHr. Rehydrated IPG gel strip is an equilibration solution containing 1% DTT (50 mM Tris, pH 8.6, 6 M urea), 30% v/v glycerol, 2% SDS. , After 15 minutes reaction in a little bromophenol blue (bromophenol blue) was reacted for 15 minutes in an equilibration solution containing 2.5% iodoacetamide (iodoacetamide) The equilibrated gel strip was made of 12.5% polyacrylamide gel. After two-dimensional electrophoresis, the protein spot is detected by silver staining The stained gel is scanned using a GS-710 densitometer and then the protein spot using PDQuest software version 6.1. After measuring the volume of the protein spot, relative to the protein isolated from the comparative E. coli, the expression level of the E. coli protein to which GdnHCl and 2-HEDS were added was compared and analyzed, and then a protein with an increased spot volume was identified.

Example 2: MALDI-TOF-MS analysis and protein identification

For the MALDI-TOF-MS analysis, a protein higher than that of the comparative E. coli selected in Example 1 was extracted from the silver-stained gel. The extracted protein spot was digested with trypsin (10.15 mg/ml) in 25 nM ammonium bicarbonate (pH 8.0) solution at 37°C overnight. The digested peptide was extracted using a 5% v/v TFA and 50% v/v ACN solution, and this process was repeated three times and then dried using a vacuum centrifuge. The dried peptide was dissolved in 50% ACN / 0.1% TFA solution and analyzed using the MALDI-TOF-MS system (Voyager DE-STR instrument; Biosystems). The analyzed peptide mass fingerprints were performed using MS-FIT (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) of the Prospector website, and MS-FIT for protein identification. Swiss-Prot was used as the database.

As a result of performing protein identification, the expression levels were increased by 1.88 times and 3.08 times or more, respectively, and structural stability was maintained under GdnHCl stress conditions, and the proteins showing stress tolerance were identified as Mdh and Tsf (Table 1).

Mdh and Tsf Protein Information
Gene name Genetic accession number ^a
Protein name Isoelectric point (pI)/molecular weight (kDa)
Sequence similarity (%) Theoretical value ^b Experimental value ^c Mdh P61889 Malate
dehydrogenase 5.61/32.34 5.63/33.12 18% Tsf P0A6P1 Elongation
factor Ts 5.22/30.29 5.15/31.62 47%

a. Gene accession number: Identification number for searching gene information from ExPASy Proteomics Server (http://www.expasy.org/)

b. Theoretical values were collected using the Compute pI/Mw tool (http://www.expasy.org/tools/pi_tool.html).

c. Experimental values were calculated from two-dimensional electrophoresis gel images.

Example 3: Preparation of an expression vector containing Mdh or Tsf at the amino terminal as a fusion partner

3-1: Expression vector containing Mdh as a fusion partner

An expression vector containing the E. coli protein Mdh (Malate dehydrogenase) having an increased water-soluble expression level under environmental stress selected by the method of Examples 1 to 2 was prepared as a fusion partner.

In order to obtain the nucleotide encoding the Mdh gene, a primer pair for PCR amplification of the Mdh gene excluding the stop codon was prepared using the sequence information (SEQ ID NO: 11) of 3381352bp to 3382290bp in gi:49175990 of the Entrez Nucleotide database. . In addition, the sense primer of the primer pair (SEQ ID NO: 1: cat atg aaa gtc gca gtc ctc ggc) has an NdeI restriction enzyme recognition sequence, and an antisense primer (SEQ ID NO: 2: ctc gag ctt att aac gaa ctc ttg) has an XhoI restriction enzyme. It was designed to include the recognition sequence. PCR is a DNA polymerase reaction buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100) ) To 100 ng of the chromosome isolated from E. coli as a template DNA, and 50 pmol of each of the primer pairs represented by SEQ ID NOs: 1 to 2, and then Taq DNA polymerase was used. Reaction conditions were carried out a total of 30 times with 95 ℃ / 30 seconds (denaturation), 52 ℃ / 30 seconds (annealing), 72 ℃ / 60 seconds (elongation), and as a result NdeI restriction enzyme to the 5 'end of the amplified DNA fragments A PCR product containing an XhoI restriction enzyme site at the site and the 3′ end was obtained. The amplified PCR products were treated with restriction enzymes NdeI and XhoI , respectively, and inserted into the restriction enzymes NdeI and XhoI sites of pT7-7 (Novagen, USA), and were named pT7-Mdh.

3-2: Expression vector containing Tsf as a fusion partner

An expression vector containing the E. coli protein Tsf (Elongation factor Ts) having an increased water-soluble expression level under environmental stress selected by the method of Examples 1 to 2 was prepared as a fusion partner.

In order to obtain the nucleotide encoding the Tsf gene, a primer pair for PCR amplification of the Tsf gene excluding the stop codon was prepared using the sequence information (SEQ ID NO: 12) of 190857bp to 191708bp in gi:49175990 of the Entrez Nucleotide database. . In addition, the sense primer of the primer pair (SEQ ID NO: 3: cat atg gct gaa att acc gca tcc ctg gta aaa) has an NdeI restriction enzyme recognition sequence, and an antisense primer (SEQ ID NO: 4: ctc gag aga ctg ctt gga cat cgc agc aac ttc) was constructed to include the XhoI restriction enzyme recognition sequence. PCR is a DNA polymerase reaction buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100) ), 100 ng of the chromosome isolated from E. coli as a template DNA and 50 pmol of each of the primer pairs represented by SEQ ID NOs: 3 to 4 were added, and then Taq DNA polymerase was used. Reaction conditions were 95°C/30 seconds (denaturation), 52°C/30 seconds (annealing), 72°C/60 seconds (kidney) for a total of 30 times. As a result, NdeI restriction enzyme at the 5'end of the amplified DNA fragment A PCR product containing an XhoI restriction enzyme site at the site and the 3′ end was obtained. The amplified PCR products were treated with restriction enzymes NdeI and XhoI , respectively, and inserted into the restriction enzymes NdeI and XhoI sites of pT7-7 (Novagen, USA), and were named pT7-Tsf.

Example 4: Preparation of ADI alone expression vector and Mdh or Tsf fusion expression vector

To confirm the induction of water-soluble expression of arginine deiminase (ADI) using Mdh or Tsf as a fusion expression partner, a fusion expression vector including ADI expression alone and a protein cleavage enzyme recognition site was prepared.

4-1: ADI's single expression vector

The target protein used in the present invention is arginine deiminase (ADI; SEQ ID NO: 13). For the template DNA of the target protein, total RNA was extracted from human tissues expressing a lot of the proteins using RNeasy mini kit (QIAGEN, USA), and then 1 µg of total RNA and 1 µl of oligo-d(T) (Invitrogen, USA, 0.5 µg/µl) was filled with distilled water up to 50 µl, and then added to AccuPower RTpremix (Bioneer, Korea) to react with RT-PCR (reverse transcription polymerasechain reaction). It was obtained by synthesizing cDNA by reacting at 70°C for 5 minutes, 4°C for 5 minutes, 42°C for 60 minutes, 94°C for 5 minutes, and 4°C for 5 minutes.

In order to prepare the single expression vector of ADI, a polymerease chain reaction was performed to include NdeI at the 5'-end and a HindIII restriction enzyme recognition site at the 3'-end of the ADI. The conditions of the polymerease chain reaction include 50 pmol each of a sense primer (SEQ ID NO: 5: ctc atg tct gta ttt gac agt) and an antisense primer (SEQ ID NO: 6: aag ctt cta tca ctt aac atc). In a buffer solution for DNA polymerase reaction (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100)) After putting 100 ng of the template DNA, PCR was performed for a total of 30 times at 95°C/30 seconds (denaturation), 52°C/30 seconds (annealing), and 72°C/60 seconds (height) using Taq DNA polymerase. Specifically, ADI amplified the amino acid sequence including the stop codon except for the start codon.

The NdeI/HindIII fragment encoding the amplified ADI was cut out and inserted into the NdeI/HindIII site of the E. coli expression vector pT7 (Novagen) vector to prepare a protein-only expression vector (Fig. 1A).

4-2: Convergence expression vector with ADI's fusion partner

A sense primer designed to include an XhoI restriction enzyme recognition sequence at the 5'-end of ADI and a HindIII restriction enzyme recognition sequence at the 3'-end (SEQ ID NO: 7: ctc gag gat gac gat gac aag tct gta ttt ga), antisense primer (SEQ ID NO: 6: aag ctt cta tca ctt aac atc) buffer solution for DNA polymerase reaction containing 50 pmol each (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl) (pH8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100), after adding 100 ng of template DNA, using Taq DNA polymerase for 95°C/30 seconds (denaturation), 52°C/30 seconds (annealing) , PCR was performed a total of 30 times at 72° C./60 seconds (height). Specifically, ADI amplified the amino acid sequence including the stop codon except for the start codon.

The PCR amplification product was treated with XhoI/HindIII restriction enzyme, and then inserted into the XhoI/HindIII site of the expression vector pT7-Mdh or pT7-Tsf prepared by the method of Example 3, thereby fusion expression vector with a fusion partner of a foreign protein. Was produced. The plasmids produced by the above method were designated as Mdh::ADI and Tsf::ADI, respectively.

In addition, glutathione-S-transferase (hereinafter'GST'), known as an effective fusion partner, was inserted into the amino terminal of ADI as a fusion partner, and GST::ADI was prepared.

4-3: expression vector for purification containing six histidine and protein cleavage enzyme sites

In order to purify ADI in which Mdh or Tsf is fusion-expressed, 6 histidines are included at the 5'-end of Mdh or Tsf, and can be recognized and cleaved by enterokinase between the Mdh or Tsf fusion partner and ADI. An expression vector for purification as follows, including the sequence, was constructed.

Sense primers (SEQ ID NO: 8: cat atg cac cat cac cat cac cat) to include 6 histidine (histidine, hereinafter'His6') and NdeI at the 5'-end of Mdh, and XhoI restriction enzyme recognition sites at the 3'-end aaa gtc gca gtc ctc ggc) and an antisense primer (SEQ ID NO: 2: ctc gag ctt att aac gaa ctc ttg) were prepared, and 6 histidines (hereinafter'His6') and NdeI, 3 at the 5'-end of Tsf '- To include the XhoI restriction enzyme recognition site at the end, a sense primer (SEQ ID NO: 9: cat atg cac cat cac cat cac cat gct gaa att acc gca tcc ctg gta aaa) and an antisense primer (SEQ ID NO: 4: ctc gag aga ctg ctt gga cat cgc agc aac ttc) was prepared. DNA polymerase reaction buffer solution containing 50 pmol each of the prepared primers (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ 20 mM Tris-HCl (pH8.8)); 2 mM MgSO ₄ 0.1 % Triton X-100), and then use Taq DNA polymerase for a total of 30 at 95°C/30 seconds (denaturation), 52°C/30 seconds (annealing), and 72°C/60 seconds (elongation). A polymerease chain reaction was performed. The amplified and purified NdeI/XhoI fragments encoding His6-Mdh and His6-Tsf were cut and inserted into the NdeI/XhoI site of the expression vector containing ADI, which were H6::Mdh::ADI and H6::Tsf. Named as ::ADI.

In addition, the 5'-end of ADI in order to separate the ADI from the fusion protein is recognized by enterokinase kinase (enterokinase) sequences that can be cut (DDDDK, less than D ₄ K) and the XhoI restriction enzyme recognition site, 3 ' to include a portion that the HindIII restriction enzyme at the terminal, the sense primer of polynucleotide fragments encoding the ADI fusion polypeptide (SEQ ID NO: 10: ctc gag gat gac gat gac aag tct gta ttt gac agt), antisense primer (SEQ ID NO: 6: aag ctt cta tca ctt aac atc) buffer solution for DNA polymerase reaction containing 50 pmol increments (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ 20 mM Tris-HCl (pH8.8); 2 mM) MgSO ₄ 0.1% Triton X-100), then add 100 ng of template DNA, then use Taq DNA polymerase for 95°C/30 seconds (denaturation), 52°C/30 seconds (annealing), 72°C/60 seconds (elongation). A total of 30 times a polymerease chain reaction was performed. After cutting the XhoI/HindIII fragment encoding the amplified enterokinase recognition sequence-target protein , it was inserted into the XhoI/HindIII site of the expression vector containing H6::Mdh and H6::Tsf, and this was H6:: It was named Mdh::EK::ADI and H6::Tsf::EK::ADI (FIG. 1B).

Example 5: Water-soluble expression of ADI recombinant protein

The ADI expression vector prepared by the method of Example 4 and the fusion expression vector with a fusion partner were transformed into E. coli and cultured, and then the expression of recombinant ADI was induced with IPTG, thereby soluble expression by Mdh or Tsf fusion partners. The effect was confirmed.

The vectors of Example 4 were transformed into E. coli by the method described by Hanahan (Hanahan D, DNA Cloning vol. 1 109-135, IRS press, 1985). Specifically, _{the vectors of Example 4 were transformed into E. coli BL21 (DE3) treated with CaCl 2} by a heat shock method, and then cultured in a medium containing ampicillin, and the expression vector was transformed to prevent ampicillin resistance. The indicated colonies were selected. E. coli transformed with the fusion expression vectors pT7-Mdh and pT7-Tsf with the fusion partner were named BL21(DE3):pT7-Mdh and BL21(DE3):pT7-Tsf. The colony was inoculated into an LB medium containing 100 mg/ml ampicillin with a portion of the seed culture medium cultured in LB medium overnight, and then incubated at 37° C. at 130 rpm. When the OD ₆₀₀ of the culture medium reached 0.5, IPTG was added (1 mM) to induce expression of the recombinant gene. After the IPTG was added, the culture was further cultured for 4 hours under the same conditions (37°C) or at 20°C at 130 rpm for 12 hours.

The E. coli cultured by the above method was centrifuged at 13,000 rpm for 5 minutes to recover the cell precipitate, and then suspended in 5 ml of a crushing solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA), and an ultrasonic disruptor (Branson sonifier, Branson Ultrasonics Corporation, USA). After crushing, centrifugation was performed at 13,000 rpm for 10 minutes, and then the supernatant and the cell lysate were separated. The cell lysate was washed twice using 1% Triton X-100. The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit (USA). The supernatant and the cell lysate were mixed with 5 X SDS (0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) and 1:4, respectively, and loaded into the wells of a 10% SDS-PAGE gel and 125 V After the sample was developed for 2 hours at, the gel was stained with the Coomassie staining method and then bleached to check the expression level of each recombinant protein with a density meter (Densitometer, Duoscan T1200, Bio-Rad, USA), and Equation 1 The solubility (%) was calculated according to.

As a result, it was confirmed that the expression of ADI fused with Mdh or Tsf was mostly higher in water-soluble expression than in insoluble aggregates, and the solubility was significantly increased in external ADI fused with Mdh or Tsf than the water-soluble expression of ADI expressed alone. It was confirmed (Fig. 2). As can be seen in FIG. 3, the level of water-soluble expression of ADI, which was expressed by fusion-expressing ADI alone and Mdh or Tsf, increased the water-soluble expression level of ADI. These results mean that Mdh or Tsf as a fusion partner has a great effect on the activity expression and production efficiency of ADI, a medical functional enzyme.

In addition, glutathione-S-transferase (hereinafter'GST'), known as an effective fusion partner, was inserted into the amino terminal of ADI as a fusion partner (GST::ADI), and E. coli BL21 ( As a result of confirming the expression pattern by the same method by transforming the strain into DE3), it was confirmed that the increase in water-soluble expression level was much more excellent when Mdh or Tsf was used as a fusion partner at the amino terminus than that using GST as a fusion partner. (Fig. 3).

Example 6: Activity of ADI recombinant protein

6-1: Purification and analysis of ADI produced through Mdh or Tsf fusion expression

Using E. coli BL21 (DE3) transformed with the expression vectors Mdh::ADI and Tsf::ADI constructed through Example 4, until the ultrasonic disruption step of Example 5 was performed in the same manner. Six histidines were located at the N-terminus of the fusion partner (Mdh, Tsf), and purification was possible through chromatography using the affinity of ^{histidin and Ni +2 metal. Ni +2} metal affinity chromatography was performed using Ni-NTA Agarose beads (QIAGEN), through which only Mdh::ADI and Tsf::ADI can be successfully purified, respectively.

In the purification process, the cells were crushed, centrifuged at 13,000 rpm for 10 minutes, and then the separated supernatant was combined with 500 mL of Ni-NTA Agarose bead (QIAGEN) for purification using metal affinity. Before reacting with Ni-NTA Agarose bead, it was washed with a binding buffer (binding buffer, pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 10 mM imidazole). The binding of the supernatant containing His6-Mdh-D4K-ADI or His6-Tsf-D4K-ADI to the Ni-NTA Agarose bead proceeds at 4°C, and after sufficiently binding for 2 hours or more, 8 mL of washing buffer [washing buffer, pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 50 mM imidazole]. The next step is a PBS buffer solution [PBS buffer, 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM sodium phosphate (Na ₂ HPO ₄₎ , 2 mM potassium monophosphate (KH ₂ PO). ₄ ), pH 7.4] was further washed once more. To His6-Mdh-D4K-ADI or His6-Tsf-D4K-ADI attached to the agarose bead, add 500 µl of terokinase solution [enterokinase 5µl, 10 X enterokinase buffer 50µl, PBS 445µl (Invitrogen)] at 22℃ After reacting for 12 hours at, the elution fraction was collected and centrifuged at 13,000 rpm for 15 minutes to separate aqueous and insoluble solutions. The separated sample was confirmed that arginine deiminease still remained in a water-soluble state even after the fusion partner Mdh or Tsf was removed through SDS-PAGE analysis (FIG. 4).

6-2: Measurement of ADI activity from which Mdh or Tsf has been removed

It is also important for the fusion expression partner to express the target protein in a water-soluble form, but it is most important to induce it to have a unique tertiary structure and express it in an active form with functionality. To confirm this, the fusion partner Mdh or Tsf and the target protein ADI were fusion-expressed, and then the fusion partner Mdh or Tsf was removed and the activity was measured.

The microbial-derived arginine deimase converts L-arginine to L-citrulline. Using these characteristics, the following analysis was performed to confirm the activity of the active ADI from which the fusion partner Mdh or Tsf obtained through Example 6-1 was removed.

100 ml of phosphate buffer (0.1 M, pH 6.5.0) and 0.1742 g L-arginine were mixed to make a 10 mM L-arginine mixture, and then 30 µl Mdh::ADI or Tsf in 270 µl of the mixed solution ::ADI supernatant and PBS buffer were added as a comparative experiment, respectively. The reaction started immediately after the addition, and the change in absorbance was measured at a wavelength of 530 nm until 12 minutes at intervals of 3 minutes after the reaction started (Duoscan T1200, Bio-Rad, Hercules, CA) (FIG. 5).

As a result, it was confirmed that there was no change in the activity of converting L-arginine to L-citrulline in the active form of ADI from which Mdh or Tsf was removed.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Preparing Active Arginine Deiminase <130> P16-B141 <160> 13 <170> KopatentIn 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mdh-S <400> 1 catatgaaag tcgcagtcct cggc 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mdh-AS <400> 2 ctcgagctta ttaacgaact cttg 24 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Tsf-S <400> 3 catatggctg aaattaccgc atccctggta aaa 33 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Tsf-AS <400> 4 ctcgagagac tgcttggaca tcgcagcaac ttc 33 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-S <400> 5 ctcatgtctg tatttgacag t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ADI-AS <400> 6 aagcttctat cacttaacat c 21 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ADI-S-XhoI <400> 7 ctcgaggatg acgatgacaa gtctgtattt ga 32 <210> 8 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Mdh-S-H6 <400> 8 catatgcacc atcaccatca ccataaagtc gcagtcctcg gc 42 <210> 9 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Tsf-S-H6 <400> 9 catatgcacc atcaccatca ccatgctgaa attaccgcat ccctggtaaa a 51 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> ADI-S-D4K <400> 10 ctcgaggatg acgatgacaa gtctgtattt gacagt 36 <210> 11 <211> 939 <212> DNA <213> Escherichia coli Mdh <400> 11 ttacttatta acgaactctt cgcccagggc gatatctttc ttcagcgtat ccagcatacc 60 ttccagcgcg ttctgttcaa atgcgctcag ggtaccgata gatttacgct cttccacgcc 120 gtttttaccc agcagcagcg gttgagagaa gaaacgggcg tactgaccgt cgccttcaac 180 gtaggcacat tcgacaacgc cttgttcgcc ctgcagtgca cgaaccagag acagaccaaa 240 acgtgcagct gcctggccca tagacagggt tgcagacccg ccaccggcct tcgcttcaac 300 cacttcagta cccgcgttct ggatgcgttt ggtcagatca gccacttcct gctcggtaaa 360 actaacgcca ggaacctgtg acagcagcgg cagaatggta acaccagagt gaccgccaat 420 aaccggcact tcaacttcgc ctggctgttt gcctttcagt tccgcaacaa aggtgttgga 480 acgaatgata tccagcgtgg taacgccgaa cagtttgttt ttgtcataaa caccggcttt 540 tttcagcact tcagcagcaa ttgcaactgt ggtgttaacc gggttagtga taataccaat 600 gcacgctttc gggcaggttt tcgcaacttg ctgtaccagg tttttcacga tgccggcgtt 660 aacgttaaac aggtcggaac gatccatacc cggtttacgc gctacgcctg cagagataag 720 aacgacatct gcgccttcca gcgccggagt cgcatcttca ccagaaaaac ctttgatttt 780 cacagcagta gggatatggc tcagatcgac agccacaccg ggagtcactg gagcgatatc 840 atacagagag agttctgaac ctgaaggcag ttgggttttt aacagtagtg caagcgcctg 900 gccaataccg ccagcagcgc cgaggactgc gactttcat 939 <210> 12 <211> 852 <212> DNA <213> Escherichia coli Tsf <400> 12 atggctgaaa ttaccgcatc cctggtaaaa gagctgcgtg agcgtactgg cgcaggcatg 60 atggattgca aaaaagcact gactgaagct aacggcgaca tcgagctggc aatcgaaaac 120 atgcgtaagt ccggtgctat taaagcagcg aaaaaagcag gcaacgttgc tgctgacggc 180 gtgatcaaaa ccaaaatcga cggcaactac ggcatcattc tggaagttaa ctgccagact 240 gacttcgttg caaaagacgc tggtttccag gcgttcgcag acaaagttct ggacgcagct 300 gttgctggca aaatcactga cgttgaagtt ctgaaagcac agttcgaaga agaacgtgtt 360 gcgctggtag cgaaaattgg tgaaaacatc aacattcgcc gcgttgctgc gctggaaggc 420 gacgttctgg gttcttatca gcacggtgcg cgtatcggcg ttctggttgc tgctaaaggc 480 gctgacgaag agctggttaa acacatcgct atgcacgttg ctgcaagcaa gccagaattc 540 atcaaaccgg aagacgtatc cgctgaagtg gtagaaaaag aataccaggt acagctggat 600 atcgcgatgc agtctggtaa gccgaaagaa atcgcagaga aaatggttga aggccgcatg 660 aagaaattca ccggcgaagt ttctctgacc ggtcagccgt tcgttatgga accaagcaaa 720 actgttggtc agctgctgaa agagcataac gctgaagtga ctggcttcat ccgcttcgaa 780 gtgggtgaag gcatcgagaa agttgagact gactttgcag cagaagttgc tgcgatgtcc 840 aagcagtctt aa 852 <210> 13 <211> 1230 <212> DNA <213> Arginine deiminase (ADI) <400> 13 atgtctgtgt ttgatagcaa atttaaagga attcacgttt attcagaaat tggtgaatta 60 gaatcagttc tagttcacga accaggacgc gaaattgact atattacacc agctagacta 120 gatgaattat tattctcagc tatcttagaa agccatgatg ctagaaaaga acacaaacaa 180 ttcgtagcag aattaaaagc aaacgacatc aatgttgttg aattaattga tttagttgct 240 gaaacatacg atttagcatc acaagaagct aaagataaat taatcgaaga atttttagaa 300 gactcagaac cagttctatc agaagaacac aaagtagttg taaggaactt cttaaaagct 360 aaaaaaacat caagagaatt agtagaaatc atgatggcag ggatcacaaa atacgattta 420 ggtatcgaag cagatcacga attaatcgtt gacccaatgc caaacctata cttcacacgt 480 gacccatttg catcagtagg taatggtgta acaatccact acatgcgtta caaagttaga 540 caacgtgaaa cattattctc aagatttgta ttctcaaatc accctaaact aattaacacc 600 ccgtggtact acgacccttc actaaaatta tcaatcgaag gtggagacgt atttatctac 660 aacaatgaca cattagtagt tggtgtttct gaaagaactg acttacaaac agttacttta 720 ttagctaaaa gcattgttgc taataaagaa tgtgaattca aacgtattgt tgcaattaac 780 gttccgaaat ggaccaactt aatgcactta gacacctggc tgaccatgtt agacaaggac 840 aaattcctat actcaccaat cgctaacgat gtatttaaat tctgggatta tgacttagta 900 aacggtggag cagaaccaca accagttgaa aacggattac ctctagaagg attattacaa 960 tcaatcatta acaaaaaacc agttctaatt cctatcgcag gtgaaggtgc ttcacaaatg 1020 gaaatcgaaa gagaaacaca cttcgatggt acaaactact tagcaattag accaggtgtt 1080 gtaattggtt actcacgtaa cgaaaaaaca aacgctgctc tagaagctgc aggcattaaa 1140 gttcttccat tccacggtaa ccaattatca ttaggtatgg gtaacgctcg ttgtatgtca 1200 atgcctttat cacgtaaaga tgttaagtgg 1230

Claims (7)

An expression vector for producing recombinant ADI comprising the Tsf gene represented by SEQ ID NO: 12 as a fusion partner with the ADI gene.
The recombinant ADI-producing expression vector according to claim 1, wherein a polynucleotide encoding a protein cleavage enzyme recognition site is ligated between the ADI gene and Tsf gene as a fusion partner.
2. The recombinant ADI-producing expression vector according to claim 1, wherein the Tsf gene as a fusion partner with the ADI gene is operably linked to a polynucleotide encoding a tag for separation and purification.
The expression vector for producing recombinant ADI according to claim 1, wherein the ADI gene is represented by SEQ ID NO: 13.
A recombinant microorganism into which the expression vector of claim 1 is introduced.
Culturing the recombinant microorganism of claim 5 to induce expression of the recombinant protein, and recovering the recombinant protein.
7. The method according to claim 6, further comprising the step of removing Tsf from the recombinant protein.

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