KR100378907B1 - Recombinant cpp-32 gene, expression vector containing the same, and method for manufacturing active cpp-32 protein in e.coli by using the same - Google Patents

Abstract

PURPOSE: Provided are recombinant CPP(cysteine putative protease)-32 gene, an expression vector containing the same, and a method for manufacturing active CPP-32 protein in E.coli by using the same, thereby simultaneously expressing p12 and p17 subunits and producing active CPP-32 in E.coli massively. CONSTITUTION: A vector pRGT7His7 CPP-32/p12-p17 contains T7 promotor, p12 coding gene, a nucleotide sequence of SEQ ID NO:5 encoding isoleucine-aspartic acid-glutamic acid-phenylalanine, and p17 coding gene. It expresses recombinant CPP-32 protein by bicistronic expression method. A transformed E.coli BL21(DE3)/pRGT7His7 CPP-32/p12-p17(KCTC-0479BP) is obtained by transformation with the above vector. A method for manufacturing the recombinant CPP-32 protein comprises culturing the transformed E.coli, followed by cytolysis, centrifuging the crude lysate to remove insoluble protein, and performing chromatography.

Description

A recombinant Cp-32 gene, an expression vector comprising the same, and a method for producing an active Cp-32 protein in Escherichia coli using the same.

The present invention relates to a recombinant gene of CPP-32 (Cysteine Putative Protease), a protein involved in apoptosis of cells, an expression vector comprising the same, and a method for producing a recombinant CPP-32 protein using the expression vector. Specifically, using the expression vector and the expression vector capable of expressing recombinant genes of p12 and p17 subunits of CPP-32 which are auto-processed upon activation by a bicistronic method. The present invention relates to a method for producing an active CPP-32 in Escherichia coli, and according to the present invention, by simultaneously co-expressing p12 and p17 monomers, CPP-32 can be easily produced in an activated form in large quantities in an individual E. coli. .

Programmed cell death, called apoptosis, plays an important role in the development of the embryo, the development of the immune system, and the maintenance of homeostasis in multicellular organisms. It is involved in cellular changes such as cell contraction, chromatin enrichment and DNA fragmentation. Active research into the mechanism of this phenomenon has recently been revealed that cancer, autoimmune diseases, and neurodegenerative diseases are related to apoptosis. (Raff, M. C (1992) Nature 356, 397-4001; Vaux, d. L., Haecher, G. and Strasser, A. (1994) Cell 76, 777-779).

To date, it is known that a group of proteases act in the process of apoptosis. The process of apoptosis in higher mammalian cells began with a study of the development of the nematode, C. elegans , and the comparison of higher biological cells with the results. In other words, CED-3, a protease that plays an essential role in cell death by driving the apoptosis of nematode C. elegans, is composed of ICE, CPP-32, a protease found in mammals, and 28% of the protein sequence. It has been reported to have identity, and studies have begun, predicting that mammalian ICE, CPP-32 proteins will play an important role in apoptosis in mammalian cells. (Yuan, J. et al. (1993) ) Cell 75, 641-652; Miura, m. Et al. (1993) Cell 75, 653-660).

CPP-32 is a new cysteine protease that has 35% protein sequence identity with CED-3 and 30% protein with ICE, more similar to CED-3 than ICE, also known as Yama or Apopain. Protein. The CPP-32 protein has a relatively short N-terminus compared to CED-3 and ICE, and lacks a peptide linker sequance present between two polypeptide units formed during autoprocessing. Compared with ICE, the amino acid residues 285 cysteine, 237 histidine, 179 arginine and 341 arginine residues, which are necessary for the binding and cleavage of this protein and substrate, are well conserved (Fernandes-Alnemri, T., Litwack). , G. and Alnemri, ES (1994) J. biol. Chem. 269, 30761-30764).

CPP-32 has two isoforms, alpha (α) and beta (β), depending on the 190th amino acid residue. The precursor (32Kd) is composed of 277 amino acid residues and is cleaved between the 28 asfamnic acid-29 serine residue and the 175 aspalic acid-176 serine residue to form P17 (29-175) and P12 (176-176). 277) (Nicholson, DW et al. (1995) Nature 376, 37-43). This process is thought to be an autoprocessing process within cells (Fernandes-Alnemri, T. et al. (1995) Cancer Res. 55, 2737-2742). On the other hand, Tewari et al. Reported that CPP-32 was processed and activated by ICE in vitro, indicating that the two proteins are closely involved in apoptosis of cells (Tewari, M. et al. (1995) Cell 81, 801-809). Apoptosis was also observed when the CPP-32 gene was overexpressed in animal or insect cells, suggesting that CPP-32 protein plays an important role in the programmed necrosis of cells. Fernandes-Alnemri, T., Litwack, G. and Alnemri, ES (1994) J. biol. Chem. 269, 30761-30764. In addition, the relationship is well known from the relationship between PARP (poly (ADR-Ribose) polymerase) protein, which cells use in response to external environmental stimuli, which is involved in DNA repair, gene conservation and monitoring. , ZQ et al. (1995) Genes Dev. 9, 509-520). 113K-sized PARP was specifically cleaved between the 216th asfamnic acid residue and the 217th glycine residue by CPP-32 at the early stage of the apoptosis process to bind the zinc-finger DNA at the N-terminus. Motif and C-terminal catalytic domains are separated (Nicholson, D. w. Et al. (1995) Nature 376, 37-43). In this case, the catalytic region of PARP cannot be located at the site where the DNA damage occurs, and the DNA can not be repaired to maintain the genome. As a result, when PARP is inactivated by CPP-32 and fails to function normally, intracellular endonuclease is activated, leading to DNA fragmentation and thus cell death (Tanaka, Y). et al. (1984) J. Biol. Chem. 259, 6579-6585). In addition, expression of the CrmA gene, a virus-derived apoptosis inhibitor, in apoptosis-producing cells did not result in the cleavage of PARP and no cell death. This is because CrmA, a specific inhibitor of ICE, is known as CPP-. It was found to inhibit the activity of 32. Furthermore, in vitro experiments did not occur when the CPP-32 activity was inhibited by treating AcPPDE-CHO in the form of Tetrapeptide aldehyde, a specific inhibitor of CPP-32. It was confirmed. These facts suggest that CPP-32 is involved in the apoptosis process in mammalian cells.

In view of the above facts, the development of a CPP-32 inhibitor that modulates the activity of CPP-32 can be used to treat apoptosis-related diseases such as cancer, autoimmune diseases and neurodegenerative diseases. His research focuses on the development of new drugs targeting 32.

Accordingly, the present inventors cloned each of the p12 and p17 monomer genes in a simple manner to produce a large amount of CPP-32 protein used to develop a CPP-32 inhibitor, etc. The present invention was completed by preparing an expression vector capable of being expressed in an individual of Escherichia coli by a toronic method and expressing it in Escherichia coli to confirm that a large amount of the active type CPP-32 protein was prepared.

It is an object of the present invention to provide a recombinant CPP-32 gene, an expression vector comprising the same, and a method for producing an active CPP-32 simply in large quantities in E. coli using the same.

Figure 1 shows the procedure for cloning the p12 and p17 monomer of the CPP-32 (Cysteine Putative Protease) protein to prepare the expression vector pRGT7His7 CPP-32 / p12-p17 of the present invention,

Figure 2 shows the changed nucleotide sequence between the p12 and p17 monomer gene of the CPP-32 protein included in the expression vector of the present invention,

Figure 3 shows the result of electrophoresis of purified recombinant CPP-32 protein by nickel affinity chromatography,

First row: 16Kda standard protein sample;

Second row: eluted fraction;

4 is a result confirming the activity of the CPP-32 protein prepared by the method of the present invention.

Line parallel to X-axis: contains only fluorescent substrate;

X-axis increasing line: contains CPP-32 protein and fluorescent substrate;

In order to achieve the above object, the present invention provides a recombinant CPP-32 gene having a changed base sequence between p12 and p17 units and its base sequence.

The present invention also provides a recombinant p12 protein comprising an isoleucine-aspam acid-glutamic acid-glutamic acid-phenylalanine amino acid sequence at the p12 protein carboxy terminus and a recombinant CPP-32 protein comprising the same and the amino acid sequence of p12.

The present invention also provides an E. coli expression vector expressing the p12 and p17 units of the recombinant CPP-32 gene by a bistronic method by the T7 agonist.

The present invention also provides an E. coli transformant that produces a recombinant CPP-32 protein transformed with the expression vector.

In another aspect, the present invention provides a method for producing a recombinant CPP-32 protein using the E. coli transformant.

Hereinafter, the present invention will be described in detail.

The present invention uses HepG2 cells derived from human hepatocytes to isolate genes encoding CPP-32 proteins involved in cell apoptosis.

Specifically, ribonucleic acid (RNA) is extracted from HepG2 cells to synthesize cDNA, and a polymerase chain reaction (hereinafter, referred to as 'PCR') is performed as a template. On the other hand, for PCR corresponding to the CPP-32 gene from the nucleotide sequence information of the CPP-32 gene (Fernandes-Alnemri, T., Litwack, G. and Alnemri, ES (1994) J. biol. Chem. 260, 30761-30764) A primer (oligonucleotide having the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4) is synthesized and used.

Specifically, the primer of SEQ ID NO: 1 includes a nucleotide sequence corresponding to the amino terminus of p12 of CPP-32 and a restriction enzyme Nde I recognition site, and a primer having a nucleotide sequence of SEQ ID NO: 2 is a restriction enzyme Cla I recognition site and a carboxy of p12. Terminal nucleotide sequences. And the primer of SEQ ID NO: 3 comprises the base sequence of the amino terminus of p17 of CPP-32 in addition to the restriction enzyme Cla I recognition site, the SD sequence (Shine-Dalgarno sequence) and the termination codon, and the primer of SEQ ID NO: 4 is a stop codon, a restriction The enzyme Xho I recognition site and the nucleotide sequence of the carboxy terminus of p17.

As a result, the p12 gene and the p17 gene containing the sequence encoding the isoleucine-aspam acid-glutamic acid-glutamic acid-phenylalanine peptide and the stop codon on the 3 'side were separated.

The expression vector pRGT7His CPP-32 / p12-p17 of the present invention is prepared by inserting the p12 and p17 genes of CPP-32 obtained in the above process into a plasmid vector pRGT7His containing a T7 agonist. The expression vector of the present invention comprises a recombinant CPP-32 gene having a changed base sequence between the p12 and p17 genes of the CPP-32 gene. Specifically, the recombinant CPP-32 gene has a nucleotide sequence of SEQ ID NO: 5 including a sequence encoding an isoleucine-aspam acid-glutamic acid-glutamic acid-phenylalanine peptide and a stop codon between p12 and p17 genes.

The expression vector pRGT7His CPP-32 / p12-p17 of the present invention was transformed into E. coli BL21 (DE3) strain, and the E. coli transformant BL21 (DE3) / pRGT7His CPP-32 / p12-p17 was transformed on May 21, 1998. It was deposited in the Gene Bank of Korea Institute of Biotechnology, Korea Institute of Science and Technology (Accession No .: KCTC-0479BP).

The present invention also provides a recombinant CPP-32 protein comprising an amino acid sequence of isoleucine-aspam acid-glutamic acid-glutamic acid-phenylalanine at the carboxy terminus of p12 protein. Specifically, the p12 protein comprises the amino acid sequence of SEQ ID NO: 6.

The present invention also provides a method for producing an active recombinant CPP-32 protein involved in apoptosis of cells. Specifically, the expression vector pRGT7His7 CPP-32 / p12-p17 is transformed into E. coli, and the recombinant CPP-32 protein of the present invention is conveniently produced in large quantities in the transformant.

In the present invention, the E. coli transformant BL21 (DE3) / pRGT7His CPP-32 / p12-p17 is sufficiently incubated, and then isopropylthio-β-D-galactoside (hereinafter referred to as “IPTG”). Is used to induce the expression of the p12 and p17 genes from the T7 effector. After culturing the transformant in which the gene expression is induced, it is crushed to obtain crude cell solution, and then centrifuged to collect only soluble proteins. A solution containing the soluble protein is passed through a nickel agarose column or the like, and the protein adsorbed on the resin is eluted to prepare a recombinant CPP-32 protein. That is, the protein expressed by the vector of the present invention contains seven histidine residues and is purified using affinity chromatography.

When the CPP-32 protein prepared by the present invention was reacted with a fluorescent substrate to find the change in fluorescence according to the degradation of the substrate, the activity of the CPP-32 protein prepared by the method of the present invention has its own properties. It was confirmed that it is a type protein.

Recombinant CPP-32 protein of the present invention can be usefully used as a search for inhibitors of CPP-32 protein, therapeutic agents for immunological disease, anticancer agents, neurodegenerative diseases and the like, which cause the production of interleukin-1β.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are merely illustrative of the invention and do not limit the scope of the invention.

Example 1 Amplification of the CPP-32 Gene.

(Step 1) cDNA synthesis from HepG2 cells.

RNA extraction from HepG2 cells was performed according to the method of Cologinski et al. (Cholozynski, P., et al. (1987) Anal. Biochem . 162, 156). 1 ml of RNA extract in 5 × 10 ⁶ cells [4M Guanidinium thiocyanate, 25 mM Sodium Citrate, pH 7.0, 0.5% Sarcosyl, 0.1M 2-mer Mercaptoethanol] was added and shaken to break the cell membrane, followed by 0.8 ml of 2M sodium acetate (pH4.0), 0.8 ml of phenol, and 1.6 ml of chloroform-isoamyl alcohol (49: 1, v / v). After mixing well and centrifuged at 12,000 g for 15 minutes at 4 ° C, the aqueous solution of the upper layer was transferred to a new container with the same volume of isopropanol, and left at -20 ° C for about 1 hour, followed by centrifugation at 12,000g at 4 ° C for 20 minutes. Isolation gave RNA precipitate. The precipitate was washed with 75% ethanol and then dried in vacuo and dissolved in 20ul of TE buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA). In order to synthesize cDNA disconnection, 10ul of the RNA solution obtained above was taken, 2ul of 0.1M methyl mercury (CH ₃ HgOH) was added and left at room temperature for 10 minutes to release the secondary structure of RNA, followed by 2ul of 1M 2-mercapto. Ethanol was added and reacted for 5 minutes. 5 μl of 10-fold concentrated reverse transcriptase reaction solution [500 mM Tris-HCl, pH8.3, 750 mM KCl, 30 mM MgCl ₂ , 10 mM Dithiothreitol], 2.5 ul 10 mM dNTP mixture (dGTP, dATP, dTTP) , dCTP 10mM), distilled water treated with 24ul diethylpyrocarbonate (DEPC), 1.0ul RNase inhibitor (RNasin, 1U / uL, Promega, USA), 1ul oligo (dT) _12-18 primer (oligo (dT) _12-18 primer, GibcoBRL, USA) was added and allowed to stand at room temperature for 10 minutes to allow RNA template and primer to adhere well, followed by 2.5ul reverse transcriptase (18U / ul, Superscript RNase H-Reverse transcriptase, BRL). , USA) was added and reacted at 37 ° C for 1 hour to synthesize cDNA strands.

(Step 2) Synthesis of the primer.

To obtain the CPP-32 gene from the cDNA obtained in step 1 using a polymerase chain reaction, the following primers were synthesized using a DNA synthesizer (DNA Synthesizer, Applied Biosystems Inc., U.S.A.).

Primer for PCR corresponding to CPP-32 gene from CPP-32 base information (Fernandes-Alnemri, T., Litwack, G. and Alnemri, ES (1994) J. biol. Chem. 260, 30761-30764) Oligonucleotides having the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, see Sequence Listing) were synthesized and used. Specifically, the primer of SEQ ID NO: 1 includes a nucleotide sequence corresponding to the amino terminus of the p12 unit of CPP-32 and a restriction enzyme Nde I recognition site, and a primer having a nucleotide sequence of SEQ ID NO: 2 is a restriction enzyme Cla I recognition site and the p12 monomer And the primer of SEQ ID NO: 3 comprises the amino terminal nucleotide sequence of the p17 unit in addition to the restriction enzyme Cla I recognition site, the SD sequence (Shine-Dalgarno sequence) and the termination codon, SEQ ID NO: 4 The primers of include the stop codon, restriction enzyme Xho I recognition site and the nucleotide sequence of the carboxy terminus of the p17 monomer.

(Step 3) Isolation of CPP-32 Gene.

To amplify the CPP-32 gene, a polymerase chain reaction was performed using a pair of primers of SEQ ID NO: 1 and SEQ ID NO: 2 and a pair of primers of SEQ ID NO: 3 and SEQ ID NO: 4. Put 2ug of each primer obtained in step 2 into the reaction tube, and add 1ng of HepG2 cell cDNA obtained in step 1 as a template, and add 10μl of 10-fold polymerization buffer solution (500mM KCl, 100mM Tris-HCl, pH 9.0, 1%). Triton X-100, 2.5mM MgCl ₂ ), 10µl of 2mM dNTP (2mM dGTP, 2mM dATP, 2mM dCTP, 2mM dTTP) 2.5 unit Taq polymerase and distilled water were added to make the total volume 100µl and then at 95 ° C. PCR was carried out 40 times under conditions of 1 minute (denatured step), 40 seconds at 55 ° C (soaking step), and 2 minutes (polymerization step) at 72 ° C. As a result, a gene of p12 unit (hereinafter referred to as "fragment p12") and a gene of p17 unit (hereinafter referred to as "fragment p17") encoding the amino acid sequence of SEQ ID NO: 6 were obtained (see sequence list).

Example 2 Preparation of an Expression Vector Comprising a Recombinant CPP-32 Gene.

2 ug of the plasmid pRGT7His7 [plasmid engineered to fuse seven histidine residues at the amino terminus of the gene inserted into the E. coli vector (Hyun-ho Chung (1993) Science 259, 806) having the T7 effector gene] 2.8 Kb nucleic acid fragments were completely cleaved with Xho I under conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothitol, pH 7.9), followed by electrophoresis with 1% agarose gel. And was named 'fragment pRGT7His7-N / X'.

Meanwhile, the fragment p12 2ug obtained in step 3 of Example 1 was subjected to the conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothitol, pH 7.9) using restriction enzymes Nde I and Cla I. After complete cleavage, the resultant was subjected to electrophoresis separation with 7% acrylamide gel to obtain nucleic acid fragments of about 350 base pairs. The fragment was named 'fragment p12-N / C'.

2ug of the fragment 'p17' obtained in step 3 of Example 1 was used as the restriction enzymes Cla I and Xho I of NEB buffer solution 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9). After complete cleavage under the conditions, electrophoresis separation was performed using 7% acrylamide gel to obtain 400 base pair nucleic acid fragments. This fragment is hereinafter referred to as 'fragment p17-C / X'.

Put 100ng fragment p12-N / C, 100ng fragment p17-C / X and fragment pRGT7His7-N / X in the conjugation reaction tube, and then 2µl of 10-fold conjugation solution (50mM Tris-HCl (pH). 7.8), 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin), 10 units of T4 DNA ligase and distilled water to a total volume of 20 μl, followed by 12 hours of reaction at 16 ° C. I was.

After the reaction, E. coli HB101 strain (ATCC 33694) was transformed to obtain an E. coli expression vector pRGT7His7 CPP-32 / p12-p17 containing the CPP-32 gene.

The nucleotide sequence of the cDNA fragment cloned into the recombinant plasmid pRGT7His7 CPP-32 / p12-p17 in the above process was stained circuit sequence according to the method of Sanger et al. (Sanger, F., et al. (1977). PNAS USA 74, 5463). It was confirmed using a determination system (Dye Cycle Sequencing System, Applied Biosystems, USA) and analyzed by ABI 377 DNA sequencer (Applied Biosystems, USA).

Selected plasmid pRGT7His7 CPP-32 / p12-p17 was transformed into an expression host E. coli BL21 (DE3) strain (Novagen Inc., Madison WI53711, U.S.A.).

Example 3 Expression of CPP-32 Gene in Escherichia Coli.

Recombinant Escherichia coli cells obtained in Example 2 were prepared using a liquid Luria medium (6% bacto tryptone, 0.5% yeast extract, 1% sodium chloride) containing 50 ug / ml of Ampicillin and 25 ug of Chloramphenicol. Shaken for 12 hours, then 3 ml of 300 ml of M9 medium (40 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8,3 mM NaCl, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM MgSO ₄ ) When the absorbance was adjusted to about 0.3 at the wavelength of 650nm, IPTG was added so that the final concentration was 0.4mM IPTG. About 4 hours after the addition of IPTG, the absorbance of the cell culture was measured, and the bacterial cell precipitates were collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman J2-21, JA14 rotor). The collected cell precipitates were confirmed by expression by electrophoresis in 15% polyacrylamide in the presence of SDS according to the method of Laemmli (Laemmli (1970) Nature 227, 680).

Example 4 Preparation of Recombinant CPP-32 Protein.

(Step 1) Cell Culture.

E. coli cells transformed in Example 3 were incubated at 37 ° C. for 12 hours, and 10 ml of the culture solution was put in 1 liter of Luria medium having the same composition as in Example 3, followed by culturing. Shake incubation at 37 ℃ for 2 hours, IPTG was added so that the final concentration of 0.4mM IPTG when the absorbance of the bacteria is about 1.0 at a wavelength of 650nm. About 4 hours after addition of IPTG, bacterial cell precipitates were collected by centrifugation at 5000 rpm for 10 minutes using a centrifuge (Beckman J2-21, JA14 rotor).

(Step 2) Cell disruption and removal of insoluble protein.

The E. coli cell precipitate expressing the recombinant CPP-32 protein obtained in the 2-liter culture was suspended by adding about 100 ml of buffer solution 1 (5 mM imidazole, 1 M NaCl, 20 mM Tris, pH 8.0) and then ultrasonically crushed in an ice bath (Ultrasonic). homogenizer 4710: Cole-Parmer, USA) and sonicated at 50% power and 50% efficiency cycle for about 2 minutes to destroy cells. The cell homogenate thus obtained was centrifuged at 10000 rpm for 30 minutes with a high-speed centrifuge (Beckman J2-21M, Rotor JA-14) to obtain a supernatant soluble protein.

(Step 3) Nickel Affinity Chromatography.

The same volume of buffer solution 1 was added to the solution of step 1 and placed in a nickel agarose column (His Bind metal chelation resin, Novagen, USA) equilibrated with buffer solution 1 and passed at a rate of 2 ml per minute. Subsequently, 50 ml of buffer 1 was flowed through the column to remove proteins that were not adsorbed on the resin. Then, 20 ml of buffer solution 1 containing 1.0 M of imidazole was added, and the proteins adsorbed on the resin were eluted at a rate of 2 ml per minute to collect 10 ml of each fraction. Collected fractions were subjected to 15% SDS-poly acrylamide gel electrophoresis (see FIG. 3 ).

Example 5 Activity measurement of the prepared CPP-32 protein.

In order to measure the enzymatic activity of the CPP-32 protein prepared in Example 4, CPP-32 (total protein; 0.5 ug), fluorescence in a buffer solution containing 50 mM HEPES (pH 7.5), 10% sncrose CHAPS, 10 mM DTT Using the substrate AcDEVD-AMC 5 uM, the reaction rate was measured over time with a fluorescence spectrometer at 25 ° C. That is, as the reaction proceeds, AcDEVD-AMC, which is a fluorescent substrate, is decomposed into AcDEVD and AMC by the activity of CPP-32, and fluorescence is increased due to the decomposition of AMC. As a fluorescence spectrometer, an AMINCO-Bowman Series 2 Luminescence Spectrometer was used, and an excitation wavelength of 350 nm and an emission wavelength of 460 nm were used. Activity measurement results are shown in FIG. 4 . From these results, it was confirmed that the purified CPP-32 protein has its own properties.

As described above, the expression vector of the present invention can simultaneously express the P12 and P17 monomer genes of CPP-32 by the bicystronic method. The CPP-32 produced by binding can be easily prepared, and in addition, the CPP-32 can be manufactured in large quantities while maintaining its own enzymatic activity.

(Sequence list)

SEQ ID NO: 1

Sequence length: 31

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 1]

(Sequence list)

SEQ ID NO: 2

Sequence length: 32

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 2]

'

(Sequence list)

SEQ ID NO: 3

Sequence length: 51

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 3]

(Sequence list)

SEQ ID NO: 4

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 4]

(Sequence list)

SEQ ID NO: 5

Sequence length: 770

Type of sequence: Nucleic acid 7

Number of chains: 1 chain

Form: Straight

Type of sequence: gene

[SEQ ID NO: 5]

(Sequence list)

SEQ ID NO: 6

Sequence length: 107

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[SEQ ID NO: 6]

Claims (3)

Comprising a T7 promoter, a gene encoding subunit p12 of CPP-32, a base sequence of SEQ ID NO: 5 encoding isoleucine-aspartic acid-glutamic acid-glutamic acid-phenylalanine and a gene encoding subunit p17 of CPP-32 , Vector pRGT7His7 CPP-32 / p12-p17 for expressing recombinant CPP-32 protein in a bicistronic manner. E. coli transformant transformed with the expression vector of claim 1 BL21 (DE3) / pRGT7 His7 CPP-32 / p12-p17 (Accession No .: KCTC-0479BP). The method of producing a recombinant CCP-32 protein, characterized in that the expression vector of claim 1 is transformed into Escherichia coli, followed by culturing and crushing to obtain crude cell solution, followed by centrifugation to separate soluble proteins, and then subjected to affinity chromatography.