KR20160088014A - 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

TECHNICAL FIELD The present invention relates to a method for preparing an active arginine deiminase,

The present invention relates to a method for producing an active arginine deiminase (ADI) using Mdh (malate dehydrogenase) or Tsf (elongation factor Ts) as a fusion partner, and more particularly, (ADI) having enhanced water solubility and folding by using an expression vector or gene construct comprising the same.

Proteins produced by biotechnology can generally be divided into medicinal and research proteins such as immunomodulatory and enzyme inhibitors and hormones and industrial proteins such as diagnostic proteins and reaction additive enzymes. Development and industrialization.

Arginine deiminase (ADI) is a bacterial enzyme that degrades arginine, a key amino acid in cancer cell metabolism, to produce citrulline and ammonia. Arginine deiminase is an enzyme that inhibits apoptosis, cancer angiogenesis, and neutralization of endotoxin through inhibition of nitric oxide synthesis, resulting in hepatocellular carcinoma, melanoma, leukemia, leukemia, prostate cancer, and the like. According to the results of previous studies, it has been known that when arginine is deprived of cancer cells with arginine deiminase, mitochondrial dysfunction, reactive oxygen species, nuclear DNA leaching and chromophore autophagy are accompanied to kill cancer cells (Chun A Changou et al., PNAS, 111 (39) 14147-14152). Due to the anticancer activities such as cancer growth inhibition and cancer angiogenesis, arginine deiminase is attracting attention as an effective enzyme for the treatment of cancer. Actually, at Polaris Pharmaceuticals in San Diego, USA, ADI-PEG20 drug was made by attaching polyethylene glycol chain having average molecular weight of 20 kDa to arginine deiminase, Has been applied to clinical trials for a variety of cancer treatments such as melanoma (Phase 2), prostate cancer, lung cancer, prostate cancer, and oral cancer (Phase I). Therefore, it is possible to use a microorganism such as Escherichia coli, which has a well-known genetic information, can apply various vector systems, and can rapidly grow at a high concentration in a relatively inexpensive medium, It is necessary to construct a system capable of producing a recombinant protein useful as a recombinant protein.

In the production of recombinant proteins in E. coli, various expression vectors with a strong inducible promoter have been developed and used in 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 E. coli, resulting in a low yield. Especially, it is known that this tendency is severe in the expression of small-sized polypeptides having a molecular weight of 10 kDa or less have. In addition, Escherichia coli generally produces insoluble aggregates in the over-expression of recombinant proteins because transcription and translation of the proteins occur at almost the same time. In the case of polypeptides expressed as aggregates, folding intermediates can interact with other protein impurities in the host cell (chaperon, ribosome, initial factor, etc.) by intermolecular disulfide bonds or hydrophobic interactions There is a drawback that the purity in the aggregate of the desired polypeptide is lowered by non-selective binding. In order to make the expressed protein active, it must be refolded by diluting with a modified product such as guanidine hydrochloride (urea) or urea (urea) (Marston FA et al., Biochem J 240 (1): 1-12, 1986).

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

In fact, several fusion partners have been studied and reported over the past several years to induce high expression of foreign proteins in E. coli (Esposito & Chatterjee, Curr Opin Biotechnol. 17: 353-358 2006; Kapust & Waugh, Protein Sci. 8: Sachdev & Chirgwin, Biochem. Biophys. Res. Commun. 244: 933-937, 1998). Maltose binding protein (MBP), Glutathione-S-transferase (GST), Thioredoxin (Trx), NusA, and the like are among the most studied fusion partners . In the case of the maltose binding protein, a wide hydrophobic gap formed by the amino acids having hydrophobicity in the portion involved in dimer formation effectively hides the hydrophobic region of the newly synthesized protein and prevents the target protein from becoming an insoluble aggregate. Bacillus is known to help the disulfide bond of the target protein, and NusA is highly active in folding when expressed in E. coli, leading to proper folding of the expressed target protein (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 65: 382-388, 1999).

Such a fusion partner has an advantage that it can easily purify a target protein expressed by fusion using an affinity chromatography method, and it is known that it helps folding of a target protein by using different molecular biological characteristics. However, these fusion partners have a disadvantage in that the yield of the target protein is remarkably lowered depending on the size of the fusion region, and the protein is not universally used for medical purposes or industrially useful proteins because it is relatively large in size compared to the target protein . In addition, there are cases in which a target protein is also produced in the form of a dimer by forming a dimer, and it is difficult to induce expression in an active form that performs a unique function even if the target protein is expressed in a water- 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 application. In addition, proteins that are commercially or medically very important are secretory proteins and membrane proteins, which are difficult-to-express proteins that produce insoluble aggregates when expressed in E. coli, have. In order to overcome this problem, it is important to develop an expression system for enhancing the expression efficiency of egg-expressing proteins by utilizing an E. coli system having various advantages, and to secure a source-based technology related thereto.

Accordingly, the present inventors have made intensive efforts to construct an active-type expression system of egg-expressing bacterial enzyme having anticancer effect. As a result, when Mdh or Tsf is used as a fusion partner to express fusion with arginine deiminase (ADI) The water-soluble and active-type expression of the arginine deiminase 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 recombinant proteins capable of improving water solubility and folding of arginine deiminase (ADI) using Mdh or Tsf gene as a fusion partner .

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

It is still another object of the present invention to provide a recombinant protein in which Mdh or Tsf produced by the above method is fused with arginine deaminase (ADI).

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

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

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

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

The present invention also provides a recombinant protein produced by the above method, wherein Mdh or Tsf and ADI are fused.

The method for producing arginine deaminase (ADI) using Mdh or Tsf according to the present invention as a fusion partner can improve the water solubility and expression ratio of egg-expressing ADI, thereby maintaining the original anticancer enzyme activity, .

1 is a schematic diagram of a fusion expression vector (A) containing only an ADI gene and a fusion expression vector (B) in which a polynucleotide encoding an enterokinase recognition site is ligated between a fusion partner (Mdh, Tsf and GST) gene and an ADI gene to be.
Figure 2 shows SDS-PAGE results of ADI-only expression and fusion partners (Mdh, Tsf and GST) and ADI fusion expression.
FIG. 3 shows the results of comparing the expression levels and aqueous expression levels of ADI-only expression and fusion partners (Mdh, Tsf and GST) with ADI fusion expression.
Figure 4 shows the results of SDS-PAGE analysis of active ADI with fusion partners (Mdh and Tsf) removed.
Figure 5 shows the activity of active ADI with fusion partners (Mdh and Tsf) removed.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In E. coli, the target protein is often expressed as an insoluble aggregate. That is, in the E. coli expression system, transcription and metastasis occur almost simultaneously, and during overexpression of the recombinant protein, the hydrophobic interaction 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 protein retains its activity even under the conditions of inhibiting the folding of the protein, and the protein having the excellent expression amount can participate in the biomechanism of E. coli to overcome the misfolding of the protein, and the protein structure is stable and self-folding ability is excellent. It can be effectively used as a partner. Maltose binding protein (MBP) (Bedouelle and Duplay, Eur J Biochem , 1998) is the most effective fusion partner of Escherichia coli fusion expression partners. However, MBP, which is known to be effective against a large number of proteins, does not exhibit an improved water-soluble expression for many proteins (Hammarstrom et al., J Struct Funct Genomics, 2006).

In the present invention, an active type of arginine deiminase (ADI) is produced by using a malate dehydrogenase (Mdh) or an enlongation factor Ts (Tsf) gene as a fusion partner to convert an insoluble aggregate into a denaturant or an activator And can maintain the original activity and structure of the enzyme without using it.

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

In the present invention, a polynucleotide coding for a protein cleavage enzyme recognition site may be connected 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 operatively linked to a polynucleotide encoding a tag for separation and purification.

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

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

In addition, the ADI gene and the Mdh gene or the Tsf gene may be operatively connected to a polynucleotide encoding a tag for separation and purification.

In the present invention, the ADI gene may be characterized by being represented by SEQ ID NO: 13.

The expression vector of the present invention can 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 sequential order.

As used herein, "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several to several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector, (C) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA. After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using a calcium chloride method or electroporation (Neumann, et al., EMBO J., 1: 841, 1982).

As a vector used for overexpression of the gene according to the present invention, an expression vector known in the art can be used. The framework vectors that may be used in the methods of the present invention include, but are not limited to, various vectors capable of transforming into E. coli selected from the group consisting of pT7, pET / Rb, pGEX, pET28a, pET-22b Can be used.

A nucleotide sequence is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide, and the promoter or enhancer is a sequence Or the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence, or the ribosome binding site is arranged to facilitate translation, Lt; / RTI > sequence. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in a selected expression host. Preferably the expression control sequence and the gene are contained within a recombinant vector containing a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the recombinant vector should further comprise a useful expression marker in the eukaryotic expression host.

The host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term "transformation" means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within 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, must also be considered.

Accordingly, the present invention provides, in a further aspect, a gene construct to which the ADI protein-encoding gene, the Mdh gene represented by SEQ ID NO: 11 or the Tsf gene represented by SEQ ID NO: 12 is linked, The present invention relates to a recombinant microorganism into which an expression vector for recombinant ADI production containing a Mdh gene represented by SEQ ID NO: 11 or a 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 the chromosome of the host cell.

It will be apparent to those skilled in the art that the present invention has the same effect as that of introducing the recombinant vector into the host cell by inserting the gene into the genome of the host cell.

In the present invention, the gene may be inserted into the chromosome of the host cell by a commonly known gene manipulation method. Examples of the method include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector , A poxvirus vector, a lentiviral vector, or a nonviral vector.

In another aspect of the present invention, there is provided a gene construct comprising a gene encoding the ADI protein, an Mdh gene represented by SEQ ID NO: 11, or a Tsf gene represented by SEQ ID NO: 12, A recombinant microorganism into which an expression vector for recombinant ADI containing the Mdh gene of SEQ ID NO: 11 or the Tsf gene of SEQ ID NO: 12 is introduced to induce the expression of the recombinant protein, ≪ RTI ID = 0.0 > ADI < / RTI >

In the present invention, it may further comprise the step of removing Mdh or Tsf from the recombinant protein.

When the objective protein to be fused with the fusion partner is an industrial protein, the fusion partner may degrade the function of the target protein, and in the case of a medical protein, the fusion partner is preferably removed since it may cause an antigen-antibody reaction. Herein, the protein cleavage enzyme recognition site may be a Xa factor recognition site, an enterokinase recognition site, a Genenase I recognition site, or a purine recognition site, or two or more of them may be used in sequence have.

In addition, a polynucleotide encoding a tag for separation purification can be operably linked to the fusion partner of the vector of the present invention or the gene of the target protein, so that the recombinant protein can be easily separated and purified. At this time, GST, poly-Arg, FLAG, poly-His and c-myc may be used singly or two or more of them may be sequentially connected. In one embodiment of the present invention, the vector of the present invention comprises a tag for separation and purification, a gene for the fusion partner, and a gene of a desired protein in a sequence in the pT7 skeletal vector, Lt; RTI ID = 0.0 > recombinant < / RTI >

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, It can be cleaved with a protein cleaving enzyme to produce the original protein of interest.

As used herein, the term "target protein" or "heterologous protein" refers to a protein that a manufacturer intends to produce in large quantities. In the present invention, a polynucleotide encoding the protein is inserted into a recombinant expression vector, Means all proteins capable of expression.

In the present invention, the term "recombinant fusion protein" means 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 produced by the above method and fused with Mdh or Tsf and ADI.

The method of producing ADI protein using Mdh or Tsf of the present invention as a fusion partner can maintain the inherent tertiary structure and inherent enzyme activity of fusion-expressed ADI, and thus can be widely used for the production of high-value-added protein drugs .

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Analysis of E. coli protein body change by environmental stress

The conditions that fusion partners should have are high expression rates and effective three-dimensional structure formation. Particularly, in order to form an effective three-dimensional structure, the structural stability of the protein must be assumed. Thus, the expression level of the production strain and the protein whose function was maintained were analyzed.

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

Escherichia coli BL21 (DE3) strain was selected for proteomic analysis, and a portion of the culture medium of the seed culture which was cultured overnight was inoculated into LB medium and cultured at 37 ° C and 130 rpm. When the culture of OD ₆₀₀ reaches 0.5, the substance of guanidine hydrochloride (guanidine hydrochloride, hereinafter 'GdnHCl') and 2-hydroxyethyl disulfide (hereinafter '2HEDS') for inhibiting the three-dimensional folding of the protein, 0.1 mM final concentration and 10 mM, and cultured for 3 hrs. The E. coli was cultured for the same time without addition of GdnHCl and 2HEDS. The cultured Escherichia coli BL21 (DE3) culture medium was centrifuged at 6000 rpm at 4 ° C, and the cells were recovered and washed twice with a pH 8.0, 40 mM Tris buffer. The washed E. coli precipitate was resuspended in 500 μl of lysis buffer (8 M urea, 4% (w / v) CHAPS, 40 mM Tris, protease inhibitor cocktail, ) And then disrupted using an ultrasonic wave crusher. After disruption of the cells, the cells were centrifuged at 12,000 rpm and 4 ° C for 60 minutes to remove cell lysate, and the supernatant was separated.

1-2: Two-dimensional gel electrophoresis for proteome 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 resuspended in a rehydration solution (2 M thiourea, 8 M urea, 4% w / v CHAPS, 1% w / v DTT, 1% w / v mobility carrier ampholyte, pH 4.7). One-dimensional isoelectric point separation was performed using a Bio-Rad Protein IEF cell electrophoresis system and an IPG (immobilized pH gradient) gel strip (17 cm, ReadyStrip) in a linear pH range of 4-7 The proteins contained in the rehydrated solution were rehydrated overnight. 2 hours at 500 V, 30 minutes at 1000 V, 30 minutes at 2000 V, 30 minutes at 4000 V, and 70000 VHr at 8000 V. The rehydrated IPG gel strips were washed with an epuilibration solution (50 mM Tris, pH 8.6, 6 M urea, 30% v / v glycerol, 2% SDS , Bromophenol blue for 15 minutes, and then reacted for 15 minutes in an equilibration solution containing 2.5% iodoacetamide. The equilibrated gel strips were applied using a 12.5% polyacrylamide gel The stained gel was scanned using a GS-710 densitometer and analyzed using a PDQuest software version 6.1 to determine the protein spot The volume of protein spot was measured and the expression level of GdnHCl and 2-HEDS-added Escherichia coli protein was compared and analyzed based on the protein isolated from the comparative E. coli. It was identified.

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

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

As a result of the protein identification, the expression levels increased by 1.88-fold and 3.08-fold in the GdnHCl stress condition, respectively, and the proteins exhibiting stress tolerance were identified as Mdh and Tsf (Table 1).

Mdh and Tsf protein information
Gene name Gene access 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. Genetic access number: An identification number for retrieving genetic information from the 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 expression vector containing Mdh or Tsf as a fusion partner at the amino terminus

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

An expression vector containing E. coli protein Mdh (Malate dehydrogenase), which has an increased water-soluble expression level under environmental stresses selected by the methods of Examples 1 and 2, as a fusion partner, was prepared.

To obtain the nucleotides encoding the Mdh gene, a pair of primers for PCR amplification of the Mdh gene except the stop codon was prepared using the sequence information (SEQ ID NO: 11) of 3381352 to 3382290 bp at 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 a NdeI restriction enzyme recognition sequence, the antisense primer (SEQ ID NO: 2: ctc gag ctt att aac gaa ctc ttg) is XhoI restriction enzyme Recognition sequence. The PCR was performed using a buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100 ), 100 ng of the template DNA and 50 pmol of each of the primer pairs shown in SEQ ID NOS: 1 to 2 were added to the chromosome from Escherichia coli and then Taq DNA polymerase was used. The reaction conditions were 30 times in total at 95 ° C for 30 seconds (denaturation), 52 ° C for 30 seconds (annealing) and 72 ° C for 60 seconds (elongation). As a result, NdeI restriction enzyme And a XhoI restriction enzyme site at the 3 ' end. The amplified PCR products were treated with restriction enzymes NdeI and XhoI , respectively, and inserted into the restriction enzymes NdeI and XhoI of pT7-7 (Novagen, USA) and named pT7-Mdh.

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

Expression vectors containing the Escherichia coli protein Tsf (elongation factor Ts), which has an increased water-soluble expression level under environmental stresses selected by the methods of Examples 1 and 2, as fusion partners were prepared.

To obtain the nucleotide encoding the Tsf gene, a pair of primers for PCR amplification of the Tsf gene except the stop codon was constructed using the sequence information (SEQ ID NO: 12) of 190857bp to 191708bp at gi: 49175990 in the Entrez Nucleotide database . The NdeI restriction enzyme recognition sequence was amplified by using an antisense primer (SEQ ID NO: 4: ctc gag aga ctg ctt gga cat cgc agc aac) in the sense primer of the above primer pair (SEQ ID NO: 3: cat atg gct gaa attacca tcc ctg gta aaa) ttc) was constructed to include the XhoI restriction enzyme recognition sequence. The PCR was performed using a buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100 ), 100 ng of the template DNA and 50 pmol of each of the primer pairs shown in SEQ ID NOS: 3 to 4 were added to the chromosomes isolated from Escherichia coli and then Taq DNA polymerase was used. The reaction conditions were 30 times in total at 95 ° C for 30 seconds (denaturation), 52 ° C for 30 seconds (annealing) and 72 ° C for 60 seconds (elongation). As a result, NdeI restriction enzyme And a XhoI restriction enzyme site at the 3 ' end. The amplified PCR products were treated with restriction enzymes NdeI and XhoI , respectively, and inserted into the restriction enzymes NdeI and XhoI of pT7-7 (Novagen, USA) and named pT7-Tsf.

Example 4: Production of ADI single expression vector and Mdh or Tsf fusion expression vector

Mdh or Tsf was used as a fusion expression partner to confirm the induction of soluble expression of arginine deaminase (ADI), a fusion expression vector containing the ADI single expression and protein cleavage enzyme recognition site was prepared.

4-1: expression vector of ADI alone

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

In order to prepare a single expression vector of the ADI, a polymerease chain reaction was performed so as to include NdeI at the 5'-end of ADI and a HindIII restriction enzyme recognition site at the 3'-end. The conditions of the polymerease chain reaction were as follows: a sense primer (SEQ ID NO: 5) and an antisense primer (SEQ ID NO: 6: aag cttcta tca ctt aac atc) the DNA polymerase reaction buffer for the solution (0.1% Triton X-100 0.25 mM dNTPs; 50 mM KCl; 10 mM (NH 4) 2 SO 4; 20 mM Tris-HCl (pH8.8);; 2 mM MgSO 4) 100 ng of template DNA was added and PCR was performed 30 times at 95 ° C for 30 seconds (denaturation), 52 ° C for 30 seconds (annealing) and 72 ° C for 60 seconds (elongation) using Taq DNA polymerase. Specifically, ADI amplified the amino acid sequence containing 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: Fusion expression vector of ADI with fusion partner

A sense primer (SEQ ID NO: 7: ctc gag gat gac gat gac aag tct gta ttt ga) prepared to include a XhoI restriction enzyme recognition sequence at the 5'-end of the ADI and a HindIII restriction enzyme recognition sequence at the 3'-end, (SEQ ID NO: 6: aag ctt cta tca ctt aac atc) a respective buffer for DNA polymerase, including reaction by 50 pmol (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 insert the template DNA 100 ng following Taq 95 ℃ / 30 seconds by using a DNA polymerase (denaturation), 52 ℃ / 30 seconds (annealing) , And 72 ° C / 60 sec (elongation). Specifically, ADI amplified the amino acid sequence containing the stop codon except for the start codon.

The PCR amplification product was treated with XhoI / HindIII restriction enzyme and inserted into the XhoI / HindIII site of the expression vector pT7-Mdh or pT7-Tsf prepared by the method of Example 3 to obtain a fusion expression vector Respectively. Plasmids prepared by the above method were named Mdh :: ADI and Tsf :: ADI, respectively.

In addition, GST :: ADI was prepared by inserting glutathione-S-transferase (GST), which is known as an effective fusion partner, as a fusion partner at the amino terminal of ADI.

4-3: Expression vector for purification comprising six histidine and protein cleavage enzyme sites

In order to purify Mdh or Tsf fusion-expressed ADI, it can be cleaved and recognized by enterokinase between Mdh or Tsf fusion partner and ADI, including 6 histidines at the 5'-end of Mdh or Tsf The following expression vector for purification containing the sequence was prepared.

(SEQ ID NO: 8) was added to the 5'-end of Mdh to contain a histidine (hereinafter, referred to as His6 ') and NdeI and a XhoI restriction enzyme recognition site at the 3'- (His6) and NdeI, 3 (SEQ ID NO: 2) were prepared at the 5'-end of Tsf , (SEQ ID NO: 9: ctc gag aga ctg (SEQ ID NO: 9)) and an antisense primer (SEQ ID NO: 9) so as to include a XhoI restriction enzyme recognition site ctt gga cat cgc agc aac ttc). 10 mM (NH ₄ ) ₂ SO _4, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO ₄ 0.1 mM, and the like were added to the DNA polymerase reaction buffer 100 ng of template DNA was added to the resulting solution at 95 ° C for 30 seconds (denaturation), 52 ° C for 30 seconds (annealing) and 72 ° C for 60 seconds (elongation) using Taq DNA polymerase Polymerease chain reaction was performed. The NdeI / XhoI fragments encoding amplified His6-Mdh and His6-Tsf were cut out and inserted into the NdeI / XhoI sites of the expression vector containing ADI, which was ligated to H6 :: Mdh :: ADI and H6 :: Tsf :: 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 10 mM (NH ₄ ) ₂ SO ₄ 20 mM Tris-HCl (pH 8.8); 2 mM (pH 7.8)) containing 50 pmol of a DNA polymerase reaction buffer (0.25 mM dNTPs; _{MgSO 4 0.1% Triton X-100} ) into the template DNA 100 ng next 95 ℃ / 30 seconds, using a Taq DNA polymerase (denaturation), 52 ℃ / 30 seconds (annealing), 72 ℃ / 60 seconds (elongation) And a total of 30 polymerease chain reactions were performed. The XhoI / HindIII fragment encoding the amplified enterokinase sequence-target protein was cut out and inserted into the XhoI / HindIII site of the expression vector containing H6 :: Mdh and H6 :: Tsf, Mdh :: EK :: ADI and H6 :: Tsf :: EK :: ADI (Fig. 1B).

Example 5: Aqueous expression of ADI recombinant protein

Expression of fusion protein with ADI single expression vector and fusion partner prepared by the method of Example 4 was transformed into Escherichia coli and the recombinant ADI expression was induced by IPTG to obtain a soluble expression by Mdh or Tsf fusion partner 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. 109-135, IRS press, 1985). Specifically, the vectors of Example 4 were transformed into Escherichia coli BL21 (DE3) treated with CaCl ₂ by a thermal shock method and then cultured in a medium containing ampicillin to transform the expression vector into ampicillin resistance Were selected. Fusion with fusion partners BLT (DE3): pT7-Mdh and BL21 (DE3): pT7-Tsf were named Escherichia coli transformed with vectors pT7-Mdh and pT7-Tsf. A portion of the culture medium in which the colonies were cultured in LB medium overnight was inoculated into LB medium containing 100 mg / ml ampicillin and cultured at 37 ° C at 130 rpm. When the OD ₆₀₀ of the culture reached 0.5, IPTG was added (1 mM) to induce the expression of the recombinant gene. After addition of IPTG, the cells were further cultured under the same conditions (37 ° C) for 4 hours or cultured at 20 ° C and 130 rpm for 12 hours.

Escherichia coli cultured by the above method was centrifuged at 13,000 rpm for 5 minutes to collect the cell pellet, suspended in 5 ml of a disruption solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA) and sonicated using a Branson sonifier Branson Ultrasonics Corporation, USA). After crushing, the supernatant was separated from the cell lysate by centrifugation at 13,000 rpm for 10 minutes. The cell lysate was washed twice with 1% Triton X-100. The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit (USA). The supernatant and cell lysate were loaded into wells of 10% SDS-PAGE gel mixed with 1: 4 with 5 x SDS (0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) , The gel was stained with Coomassie staining method and decolorized. The expression amounts of the respective recombinant proteins were confirmed with a density meter (Densitometer, Duox T1200, Bio-Rad, USA) The solubility (%) was calculated according to the following formula.

As a result, the expression of ADI fused with Mdh or Tsf was found to be higher in water-soluble expression than that of insoluble aggregate, and the solubility was significantly increased in the other ADIs fused with Mdh or Tsf than the soluble expression amount of ADI alone (Fig. 2). As shown in FIG. 3, the water-soluble expression level of ADI that fused single-expressed ADI with Mdh or Tsf was increased. These results indicate that Mdh or Tsf as a fusion partner is effective for increasing the expression and activity of ADI, a medical functional enzyme.

In addition, glutathione-S-transferase (GST), which is known to be an effective fusion partner, was inserted into the amino terminus of ADI as a fusion partner (GST :: ADI) and E. coli BL21 DE3) and confirmed the expression pattern by the same method. As a result, it was confirmed that the use of Mdh or Tsf as a fusion partner at the amino terminus was much better than that of 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

The same procedure was repeated up to the ultrasonic disruption step of Example 5 using the expression vector Mdh :: ADI constructed through Example 4 and Escherichia coli BL21 (DE3) transformed with Tsf :: ADI. Six histidine residues were located at the N - terminus of the fusion partner (Mdh, Tsf), and purification was possible by chromatography using affinity of histidine and Ni ^{+ 2} metal. Ni ^{+ 2} metal affinity chromatography was performed using Ni-NTA Agarose bead (QIAGEN), whereby only Mdh :: ADI and Tsf :: ADI can be successfully purified, respectively.

After the cells were disrupted, the cells were centrifuged at 13,000 rpm for 10 minutes, and the separated supernatant was combined with 500 mL of Ni-NTA agarose bead (QIAGEN) for purification using metal affinity. Prior to reaction with Ni-NTA agarose beads, they were washed with binding buffer (pH 8.0, 50 mM sodium phosphate, 300 mM 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 proceeded at 4 ° C and allowed to bind for more than 2 hours, then washed with 8 mL of washing buffer pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 50 mM imidazole]. In the next step, PBS buffer, 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM potassium phosphate KH ₂ PO ₄ ), pH 7.4]. 500 μl of enterokinase solution (50 μl of enterokinase, 10 μl enterokinase buffer, 445 μl of PBS (Invitrogen)] was added to His6-Mdh-D4K-ADI or His6-Tsf-D4K-ADI attached to agarose bead at 22 ° C For 12 hours. The elution fraction was taken and centrifuged at 13,000 rpm for 15 minutes to separate the water-soluble and insoluble solution. SDS-PAGE analysis of the separated samples confirmed that the arginine deiminase still remained in a water-soluble state after the fusion partner Mdh or Tsf was removed (FIG. 4).

6-2: Measurement of activity of ADI with Mdh or Tsf removed

It is important that the fusion expression partner is expressed in a water-soluble form, but it is important to induce the fusion protein to have an inherent tertiary structure and to express it in an active form having a functional property. To confirm this, the fusion partner Mdh or Tsf and the target protein ADI were fused and expressed, and then the fusion partner Mdh or Tsf was removed and the activity was measured.

The microorganism-derived arginine deiminase enzyme converts L-arginine to L-citrulline. Using these characteristics, the following assay was performed to confirm the activity of the active ADI with the fusion partner Mdh or Tsf removed through Example 6-1.

Arginine was mixed with 100 ml of phosphate buffer (0.1 M, pH 6.5.0) and 0.1742 g of L-arginine, and 30 μl of Mdh :: ADI or Tsf :: PBS buffer was added as a comparative experiment with the ADI supernatant. The reaction was started immediately after addition and the change in absorbance at 530 nm wavelength was measured (Duoscan T1200, Bio-Rad, Hercules, Calif.) (Figure 5).

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

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Preparing Active Arginine Deiminase <130> P15-B011 <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 (12)

A gene construct to which an ADI protein-encoding gene, an Mdh gene represented by SEQ ID NO: 11, or a Tsf gene represented by SEQ ID NO: 12 is linked.
The gene construct according to claim 1, wherein a polynucleotide encoding a protein cleavage enzyme recognition site is linked between the ADI gene and the Mdh gene or the Tsf gene.
The gene construct according to claim 1, wherein the ADI gene and the Mdh gene or the Tsf gene are operably linked to a polynucleotide encoding a tag for separation and purification.
An expression vector for producing recombinant ADI comprising the Mdh gene represented by SEQ ID NO: 11 as the fusion partner with the ADI gene or the Tsf gene represented by SEQ ID NO: 12.
The recombinant ADI-producing expression vector according to claim 4, wherein a polynucleotide encoding a protein cleavage enzyme recognition site is linked between the ADI gene and a Mdh gene or a Tsf gene as a fusion partner.
5. The expression vector for recombinant ADI production according to claim 4, wherein the Mdh gene or the Tsf gene as a fusion partner with the ADI gene is operably linked to a polynucleotide encoding a tag for separation and purification.
5. The recombinant ADI-producing expression vector according to claim 4, wherein the ADI gene is represented by SEQ ID NO: 13.
A recombinant microorganism into which the gene construct of claim 1 or the expression vector of claim 4 has been introduced.
9. The recombinant microorganism according to claim 8, wherein the gene construct is inserted into the chromosome of the host cell.
Culturing the recombinant microorganism of claim 8 to induce expression of the recombinant protein, and recovering the recombinant protein.
11. The method according to claim 10, further comprising the step of removing Mdh or Tsf from the recombinant protein.
10. A recombinant protein produced by the method of claim 10, wherein Mdh or Tsf and ADI are fused.

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