KR101717238B1 - Recombinant microorganism producing Crotamine protein and method for Production of Crotamine protein Using Thereof - Google Patents

Abstract

The present invention relates to a recombinant vector for producing a crotamine protein, a recombinant microorganism producing the crotamine protein transformed with the vector, and a method for producing the crotamine protein using the recombinant microorganism. In the recombinant microorganism transformed using the recombinant vector for producing a crotamine protein containing the tag protein of the present invention, the crotamine is normally folded and overexpressed by water, so that the crotamine protein can be mass produced, The crotamine protein produced by the method can be used as a composition for anticancer, antibacterial or antifungal; Active proliferating cell markers; And markers of the central and cell cycle.

Description

TECHNICAL FIELD The present invention relates to a recombinant microorganism producing a crotamine protein and a production method of the crotamine protein using the recombinant microorganism.

The present invention relates to a recombinant vector for producing a crotamine protein, a recombinant microorganism producing the crotamine protein transformed with the vector, and a method for producing the crotamine protein using the recombinant microorganism.

Crotamine (Crt) is a 5 kDa peptide toxin found in the venom of the South American rattlesnake ( Crotalusdurissusterrificus ) [Laure CJ, 1975, Hoppe Seylers Z Physiol Chem 356: 213-215.], Giglio JR 1975, Anal Biochem 69: 207-221). Crotamine belongs to the myneurotoxin protein family, which includes skeletal muscle spasms with negligible toxicity (LD ₅₀ = 33 μg / g) when compared to members of other families [Nicastro G, Franzoni L, de Chiara C, Mancin AC, Giglio JR, et al., (2003), Eur J Biochem 270: 1969-1979]. The initial voltage-gated calcium channel was considered to be a molecular target of crotamine, but it has been suggested that the initial voltage-dependent calcium channel is a molecular target of crotamine, (1999), Toxicon 50 (1999), and Eli J Pharmacol 348: 167-173), which have been controversial [Rizzi CT, Carvalho-de-Souza JL, Schiavon E, Cassola AC : 553-562]. The voltage-gated K (Kv) pathway was considered to be a target of crotamine based on computational docking (Yount NY, Kupferwasser D, Spisnia A, Dutz SM, Ramjan ZH, et al. , (2009), Proc Natl Acad Sci USA 106: 14972-14977]. Recently, it has been confirmed that crotamine selectively inhibits Kv1.1, Kv1.2 and Kv1.3 channels with an IC ₅₀ of up to 300 nM, but not other K channels [Peigneur S, Orts DJ , Prieto da Silva AR, Oguiura N, Boni-Mitake M, et al., (2012), Mol Pharmacol 82: 90-96]. In addition, crotamine showed the possibility of anticancer activity and induces apoptosis in a concentration dependent manner on proliferating cells such as various tumor cells [Kerkis A, Kerkis I, Radis-Baptista G, Oliveira EB, Vianna-Morgante AM, et al., (2004), FASEB J 18: 1407-1409]. Crotamine has also been shown to be effective in mouse models of melanoma [Oguiura N, Boni-Mitake M, Affonso R, Zhang G, (2011) J Antibiot (Tokyo) 64: 327-331], [Nascimento FD , Sancey L, Pereira A, Rome C, Oliveira V, et al., (2012), Mol Pharm 9: 211-221]. In addition, strong activity against Gram-positive yeast Candida spp. (Gram-positive and Gram-negative bacteria) showed some activity, and crotamine was found to have antibacterial and antifungal activity [Yamane ES, Bizerra FC, Oliveira EB, Moreira J, Rajabi M, et al., (2013), Biochimie 95: 231-240]. In addition, crotamine has properties such as cell penetration activity, DNA binding ability, blood brain barrier passing ability, and the like; active proliferation cell markers in vitro and in vivo; And as markers of the central and cell cycle. Because of this potential biomedical application, the mass production of crotamine is of value.

Crotamine is a native form and has been purified from the poison of South American rattlesnakes, but it is not suitable for mass production, and purity purity may be a problem. Also, when overexpression in eukaryotes is used, there is a problem because crotamine has cell-permeability and cytotoxicity.

Therefore, the inventors of the present invention have been studying a method for mass-producing crotamine efficiently, and a recombinant vector containing a tag protein is prepared, and crotamine is normally folded in E. coli transformed with the vector, The present inventors have completed the present invention by confirming that crocamin can be efficiently mass produced.

It is an object of the present invention to provide a recombinant vector for production of a crotamine protein.

It is also an object of the present invention to provide a recombinant microorganism producing a crotamine protein transformed with the recombinant vector for producing the crotamine protein.

It is another object of the present invention to provide a method for producing a crotamine protein using the recombinant microorganism.

In order to solve the above problems, the present invention provides a recombinant vector for producing a crotamine protein, which comprises a DNA encoding a tag protein and a DNA encoding a crotamine.

The present invention also provides a recombinant microorganism producing a crotamine protein transformed with the recombinant vector for producing the crotamine protein.

The present invention also provides a method for producing a recombinant microorganism, comprising: 1) culturing the recombinant microorganism; And 2) separating and purifying the chromatin protein from the culture broth.

In the recombinant microorganism transformed using the recombinant vector for producing a crotamine protein containing the tag protein of the present invention, the crotamine is normally folded and overexpressed by water, so that the crotamine protein can be mass produced, The crotamine protein produced by the method can be used as a composition for anticancer, antibacterial or antifungal; Active proliferating cell markers; And markers of the central and cell cycle.

1 is a diagram showing a vector map of pHMGWA-MBP-TEVrs-Crt.
Figure 2 shows the approximate structure of His6-, Trx-, GST-, PDIb'a'-, MBP-, PDI- and NusA-crotamine (Crt) fusion proteins.
FIG. 3 shows the results of analysis of the expression of crotamine fused with seven different tag proteins in recombinant E. coli using SDS-PAGE (A: 37 ° C, B: 20 ° C)
4 is a diagram showing a purification process of a crotamine protein in a recombinant E. coli.
FIG. 5 is a graph showing SDS-PAGE of the purified crotamine protein in recombinant E. coli (M: molecular weight marker, lane 1: total cells before IPTG induction (negative control), lane 2: total cells treated with IPTG, Lane 3: MBP tag (40.2 kDa) cleavage using purified water-soluble fraction, lane 4: HisTrap FF column, purified MBP-Crt fusion protein (48.9 kDa), lane 5: TEV protease (29 kDa) Lane 6: final purified crotamine (5 kDa)).
FIG. 6 is a graph showing the results of confirming the purified crotamine protein using the silver-stained gel. FIG.
FIG. 7 is a diagram showing a trypsin-degrading peptide map (43 amino acids) of a crotamine protein.
FIG. 8 is a graph showing the results of MALDI-TOF MS analysis of the purified crotamine protein under reducing conditions. FIG.
FIG. 9 is a diagram showing the results of MALDI-TOF MS analysis of purified crotamine protein under non-reducing conditions. FIG.
Figure 10 shows superimposed current traces according to the concentration of chromatin protein (30 pM, 300 pM, 3 nM, 30 nM, 300 nM and 3 uM) on Kv1.3 channel current in frog oocytes .
11 is a graph showing a current-voltage relationship curve of a 3 nM concentration of a crotamine protein or a control group for Kv1.3 channel current in frog oocytes.
12 is a graph showing the inhibitory effect of chromatin protein on Kv1.3 channel current in frog oocytes according to the concentration of crotamine.
13 is a diagram showing superimposed current traces according to the concentration of chromatin protein for Kv1.5 channel current in frog oocytes.
14 is a graph showing a current-voltage relationship curve of a 3 nM concentration of a crotamine protein or a control group for a Kv1.5 channel current in frog oocytes.

The present invention provides a recombinant vector for producing a crotamine protein, which comprises DNA encoding a tag protein and DNA encoding crotamine.

For purposes of the present invention, a "vector" means a DNA product that retains 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 can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.

The term "expression vector" as used herein refers to a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include, but are not limited to, antibiotic resistance genes such as ampicilin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Selectable.

The tag proteins include maltose-binding protein (MBP), hexahistidine, Trx (thioredoxin), glutathione S-transferase (GST), protein disulfide isomerase b'a 'domain, isomerase and NusA (N-utilizing substance protein A), but the present invention is not limited thereto.

The His6 protein is SEQ ID NO: 3, the Trx protein is SEQ ID NO: 4, the GST protein is SEQ ID NO: 5, the PDIb'a 'is SEQ ID NO: 6, the PDI is SEQ ID NO: 7, The NusA protein may comprise, but is not limited to, the amino acid sequence shown in SEQ ID NO: 8.

The DNA encoding the tag protein contained in the recombinant vector includes all nucleotide sequences capable of encoding the respective tag proteins, but is not limited thereto.

The DNA encoding the crotamine includes all of the nucleotide sequences capable of encoding the amino acid sequence shown in SEQ ID NO: 1, but is not limited thereto.

In the tag and crotamine proteins of the present invention, not only a protein having a naturally occurring amino acid sequence but also amino acid sequence variants thereof are included within the scope of the present invention. A tag protein and a variant of a crotamine protein means a protein having a sequence different from that of a tag protein and a crotamine protein by a deletion, insertion, non-conservative or conservative substitution or a combination thereof with a natural amino acid sequence and one or more amino acid residues. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art. The tag protein and the crotamine protein; Or variants thereof can be prepared by natural recombinant methods, such as those derived from natural sources or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or DNA sequences (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989).

In the present invention, the gene capable of coding the tag protein and the crotamine protein can be synthesized artificially using a nucleic acid synthesizer or the like, or genomic DNA of a gene capable of encoding a tag protein and a crotamine protein as a template, PCR using oligonucleotide having a sequence complementary to both ends of a gene capable of encoding a tag protein and a crotamine protein as a primer. On the other hand, due to the optimization of codons, the genes capable of coding the tag and crotamine proteins of the present invention can exist in various base sequences, all of which are included in the scope of the present invention. Variants of the above base sequences are also included in the scope of the present invention. Specifically, a gene capable of encoding a tag protein and a crotamine protein has a homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% Base sequence. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The recombinant vector may include DNA encoding a protease recognition site. The protease recognition site is included to easily isolate the crotamine protein from the expressed tag protein-crotamine fusion protein. The protease includes the factor Xa protease, thrombin, enterokinase and TEV (Tavacco Etch Virus ) Protease, but is preferably TEV (Tavacco Etch Virus) protease, but is not limited thereto. Since the TEV protease recognizes the ENLYFQ ↓ G (Glu-Asn-Leu-Tyr-Phe-Gln ↓ Gly) sequence and cleaves the marker site, when the DNA encoding the sequence is inserted into the vector, The crotamine protein can easily be recovered from the fusion protein.

The recombination vector may have a cleavage map as shown in the following figures, but is not limited thereto.

[drawing]

The present invention also provides a recombinant microorganism producing a crotamine protein transformed with the recombinant vector for producing the crotamine protein.

The recombinant microorganism may be at least one species selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, preferably E. coli, but is not limited thereto.

The method of transforming the microorganism with the recombinant vector may be carried out by conventional transformation methods such as cationic lipid-mediated transfection, electroporation, DEAE dextran mediated transformation transfection, and calcium phosphate transfection. Other optimal transformation methods may be introduced depending on the kind of the recombinant microorganism and expression vector with reference to what is known in the art.

The present invention also provides a method for producing a recombinant microorganism, comprising: 1) culturing the recombinant microorganism; And 2) separating and purifying the chromatin protein from the culture broth.

In the step 1), "cultivation" means growing the microorganism under moderately artificially controlled environmental conditions.

The recombinant microorganism can be grown in a conventional medium, for example, in a nutrient broth medium. The culture medium may contain nutrients required for culturing, that is, a microorganism to be cultured in order to cultivate a specific microorganism, and may be a mixture in which a substance for a special purpose is further added and mixed. The medium is also referred to as an incubator or a culture medium, and is a concept including both natural medium, synthetic medium and selective medium. The recombinant microorganism can be cultured according to a conventional culture method.

The medium used for cultivation should meet the requirements of a specific strain in a suitable manner while controlling temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.

In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.

The recombinant microorganism in step 1) may be cultured at 15 to 40 ° C, preferably at 18 to 38 ° C, but is not limited thereto.

The separation and purification of the crotamine protein from the culture broth in the step 2) can be carried out using a conventional protein separation method. For example, the crotamine protein may be isolated from the culture medium by methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Further, the crotamine protein can be purified through known methods including chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (e.g., ammonium sulfate precipitation), SDS- .

In one embodiment of the present invention, the culture of the recombinant microorganism was sequentially treated with Immobilized Metal Affinity Chromatography (IMAC), TEV protease treatment, and cation exchange chromatography to finally obtain a pure crotamine protein. The crotamine protein according to the above method obtained 0.90 mg of pure crotamine per 1 L cell culture medium, and the crotamine protein is composed of the amino acid sequence shown in SEQ ID NO: 9.

In the recombinant microorganism transformed with the recombinant vector for producing crotamine protein containing the tag protein of the present invention, crotamine is normally folded and overexpressed by water, so that crotamine can be mass produced using the same.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1. Preparation of recombinant plasmid for production of crotamine protein

The attB1-TEVrs-Crt-attB2 gene was inserted into the pUC57 vector containing the EcoRV cloning site using the bioneer gene synthesis service to synthesize a DNA sequence encoding 42 amino acids of crotamine. Specifically, the site of attB1 (5'-ggggacaagtttgtacaaaaaagcaggcttc-3 ', SEQ ID NO: 10) and attB2 (5'-acccagctttcttgtacaaagtggtcccc-3', SEQ ID NO: 11) were ligated to the 5'- and 3'- Lt; / RTI > Subsequently, tobacco etch virus recognition site (TEVrs; ENLYFQ ↓ G) was inserted between the attB1 site and the crotamine site for cleavage of the tag protein with crotamine (SEQ ID NO: 1) . Subsequently, the whole vector named pENTR-TEVrs-Crt was prepared by subcloning the DNA synthesized by BP recombination reaction (Invitrogen, Carlsbad, Calif.) Into pDONOR207 vector. Then TEVrs-Crt of pENTR-TEVrs-Crt was ligated to each MBP (SEQ ID NO: 2), His6 (SEQ ID NO: 3), Trx (SEQ ID NO: 4), GST (SEQ ID NO: HGWA, pDEST-HGGWA, pDEST-HNGWA, pDEST-HNGWA, pDEST-HGGWA encoding the tag proteins of hPDIb'a (SEQ ID NO: 5), hPDIb'a -HXGWA, pDEST-hPDI and pDEST-hPDIb'a 'destination vectors. Sequences prepared by the BP and LR reactions were confirmed using a DNA sequencing service of Macrogen (Daejon, Korea). The His6 tag and the His8 tag (hPDIb'a 'and hPDI) were included in front of each tag protein for ease of purification.

Seven expression plasmids were obtained by over-expressing recombinant crotamine fused with His6-, Trx-, GST-, hPDIb'a'-, MBP-, hPDI- and NusA-tag proteins by a gateway system. attP1 and attP2 of the pDONR207 vector reacted with the TEVrs-Crt gene in the pUC57-Crt vector and reacted with the attB1 and attB2 of the pUC57-Crt vector, which exchanges the ccdB and Cm genes of the pDONR207 vector. As a result of the above reaction, pENTR-Crt produced from attL1-TEVrs-Crt-attL2 was produced. Then, attL1 and attL2 reacted with attR1 and attR2 in the respective pDEST-HGWA, pDEST-HGGWA, pDEST-HMGWA, pDEST-HNGWA, pDEST-HXGWA, pDEST-hPDI, and pDEST-hPDIb'a ' . In this step, ccdB and Cm (R) in each destination vector were exchanged for TEVrs-Crt. Finally, each of the seven expression plasmids regulated by the T7 promoter was produced between the MBP-fused structures, and the vector production process for producing the MBP-tagged protein-crotamine was shown in FIG. In addition, the approximate structure of each of the seven tag proteins and the crotamine protein is shown in Fig.

Example 2 Expression and Solubility of Recombinant Crotamine according to Temperature Conditions

To confirm the expression and solubility of each of the seven tag-protein-recombinant cortamines prepared in Example 1 according to the culture temperature conditions (37 ° C and 20 ° C), the following procedure was performed.

More specifically, competent E. coli BL21 (DE3) was transformed using an expression vector. The transformant was transformed into Luria-Bertani medium containing 50 μg / mL ampicillin at 37 ° C. and A ₆₀₀ optical And incubated until reaching optical density. Expression system for 3 hours at 37 < 0 >C; And 0.5 mM isopropyl -? - D-thiogalactoside (IPTG) at 20 ° C for 18 hours. The recombinant E. coli culture was sonicated with Tris buffer and the aqueous and non-aqueous portions were centrifuged at 18,000 g, 15 min and 4 ° C. The expression and solubility of recombinant chromatin were analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using a 10% tris-tricine gel gel. ImageJ program ( http: //imagej.nih. gov / ij ). The results are shown in FIG. 3 and Table 1.

Protein Tag Tag size
(kDa) Fusion size
(kDa) Expression level (%) Solubility (%) 37 ^o C 20 ^o C 37 ^o C 20 ^o C Crotamine
(5 kDa) His6- 0.8 8.6 18.0 9.6 56.5 37.9 Trx- 11.7 20.4 38.9 28.8 53.1 50.7 GST- 25.6 34.3 20.7 22.0 63.2 69.5 PDIb'a'- 30.6 39.6 38.4 50.6 75.2 89.1 MBP- 40.2 48.9 54.1 65.8 63.0 71.2 PDI- 54.8 63.8 28.7 30.4 70.3 67.4 NusA- 54.7 63.5 37.2 53.3 82.0 80.0

As shown in FIG. 3 and Table 1, when the recombinant E. coli transformed with the four recombinant vectors containing Trx, hPDIb'a ', MBP and NusA was cultured at 37 ° C, a high expression level of 30% or more Respectively. It was confirmed that expression levels were increased at low temperatures in the recombinant E. coli transformed with the recombinant vector containing the five tag proteins other than the recombinant vector having the His6- and Trx-tagged proteins. Recombinant E. coli transformed with the MBP-tagged crotamine-containing recombinant vector showed 54.1% and 65.8% of the highest expression levels at 37 ° C and 20 ° C, respectively, than the rest of the tagged proteins.

At 20 ° C, the solubility of the remaining tagged protein-crotamine was confirmed to be 50% or more, except for His6-tagged crotamine, and the solubility of all the tagged proteins-crotamine was found to be more than 50% at 37 ° C .

It was also confirmed that MBP-tagged crotamine and NusA-tagged crotamine exhibited the highest expression level and solubility.

Example 3. Purification of crotamine

In order to purify and efficiently obtain a crotamine protein, the following experiment was conducted.

More specifically, the cell pellet obtained by centrifugation of the recombinant E. coli cells cultured at 20 占 폚 for 18 hours at 3,600 g for 30 minutes at 4 占 폚 was cooled to -20 占 폚. The cooled cell pellet was resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% Glycerol (v / v), 5 mM mercaptoethanol) at a rate of 12.5 mL / , The lysate was sonicated 25 times with on / off of 20 sec / 10 sec. The water-soluble protein of the treated supernatant was obtained by centrifugation at 34,500 g for 10 minutes at 4 ° C. After filtration using a 0.45 μm syringe filter (Chemcokorea, Seoul, Korea) Respectively.

The filtered supernatant was applied to a HisTrap FF affinity column (GE Healthcare, Piscataway, NJ) equilibrated with dissolution buffer using a fast protein liquid chromatography system. The column was washed with 40 column volumes (CV) of buffer A (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% glycerol (v / v) - mercaptoethanol, 50 mM imidazole). The elution step was then carried out using buffer B (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% glycerol (v / v), 5 mM [beta] -mercaptoethanol, 500 mM imidazole). A 14 kDa cut-off membrane (Viskase Corporation, Darien, IL) was coated with buffer C (20 mM Tris-HCl pH 8.0, 5% glycerol (v / v), 100 mM NaCl, Purified TEV was mixed at 25 rpm and 85 rpm at a ratio of 1:10 (TEV: fusion protein; w / w) for 24 hours to obtain a sample solution of MBP-Crt Lt; / RTI >

After the TEV treatment, the precipitate was removed by centrifugation at 1,500 g for 20 minutes at 4 ° C, and a supernatant was obtained. The TEV-treated supernatant sample was concentrated to 5-10 ml and dialyzed at room temperature for 5 hours using a dialysis membrane containing 3,500 Da molecular cutoff (Viskase, Darien, IL) (50 mM MES pH 6.0, 5% glycerol (v / v), 2 mM DTT). The dialyzed sample was filtered through a 0.45 μm syringe filter (Chemcokorea, Seoul, Korea) prior to application of the equilibrated sample in Buffer D to 5 mL of HiTrap SP HP (GE healthcare, Piscataway, NJ).

To remove impurities, a gradient (0-400 mM) of NaCl concentration was used. The cleaved crotamine was dissolved in 5 CVs of buffer E (50 mM MES pH 6.0, 5% Glycerol (v / v), 2 mM DTT, 1 M NaCl). A 5 mL MBP Trap HP column (GE healthcare, Piscataway, NJ) equilibrated with buffer F (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% Glycerol Column. The MBP flow through the cleaved containing pure crotamine was changed to PBS by dialysis. All purified fractions were analyzed by SDS-PAGE with 10% tris-tricine gel. The purified crotamine protein concentration was measured using a BCA protein assay (Pierce Biotechnology, Rockford, IL) with BSA as the standard (Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, et al. (1985), Anal Biochem 150: 76-85]. As described above, the process for purifying crotamine from recombinant E. coli is shown in Fig. 4, and the purified crotamine is represented by the amino acid sequence shown in SEQ ID NO: 9.

Example 4. Analysis of purified crotamine by electrophoresis and silver staining

In order to analyze the purified crotamine protein in Example 3 using electrophoresis and silver staining, the following experiment was conducted.

More specifically, SDS-PAGE was performed using the method of Laemmli UK (1970), and samples of each step in Example 3 were diluted with sample buffer (5X: 200 mM Tris-HCl, pH 6.8, 48% glycerol, 16% SDS, 0.04% Coomassie blue G-250, 0.4 M DTT) for 10 minutes at 100 ° C. It was then loaded onto a 10% polyacrylamide gel. Protein bands were identified by staining with Coomassie brilliant blue R-250 (AMRESCO, Solon, Ohio) and analyzed with Image J image analysis software ( http://imagej.nih.gov/ij ) 5).

In addition, the final purified crotamine was identified using the Silver Stain Plus kit (Bio-Rad Laboratories, Hercules, Calif.) According to the manufacturer's protocol. When the band appeared clear, the reaction was stopped by treating with 5% acetic acid (v / v) for 15 minutes. The reacted gel was thoroughly washed with water and kept overnight in an aqueous solution containing 5% glycerol (v / v) and 0.02% sodium azide (w / v) (see FIG. 6). The results are shown in FIGS. 5 and 6. FIG.

As shown in FIG. 5, it was confirmed that MBP-tagged crotamine containing His6 at the N-terminus was purified by Immobilized Metal Affinity Chromatography (IMAC) containing nickel containing two main impurities (lane 4). It was also confirmed that after the MBP column, 0.90 mg of pure crotamine was obtained per 1 L cell culture (lane 6).

Further, as shown in Fig. 6, it was confirmed that a very high purity chromatin was obtained.

Example 5. MALDI-TOF MS analysis for disulfide bond analysis of crotamine

For the presence or absence of the disulfide bond of the purified recombinant crotamine obtained in Example 3, the following experiment was carried out using samples of reduction and non-reduction conditions depending on the presence or absence of dithiothreitol (DTT) Respectively.

More specifically, FIG. 7 shows a tryptic digesting peptide map of crotamine represented by crotamine (43 amino acids) digested by trypsin treatment. For the reducing conditions, 2 ㎍ recombinant crotamine was treated with 10 mM DTT for 15 minutes at room temperature, and then the solution was evaporated by a vacuum drying method. The precipitate was then washed with IAA buffer (0.5 M Tris-HCl, pH 8.0, 5% glycerol (v / v), 100 mM NaCl, 1 mM EDTA and 2% SDS ), And then cultured for 1 hour at room temperature. The cultured sample was loaded on 10% tris-tricine gel, and the band corresponding to the recombinant crotamine was cut off and treated with trypsin. MALDI-TOF was performed using Voyager-DE STR (Applied Biosystems, South San Francisco, Calif.) And data analysis was performed using Data Explorer software (Applied Biosystems). The mass of the obtained peptide was determined using Peptide Mass program ( http://web.expasy.org/peptide_mass ). The results are shown in Fig. 8 and Fig.

T8 + IAA, T4 + IAA, and T6 + IAA, as shown in Fig. 8, under most of the major fragments including the alkylated cysteine and free cysteine IAA). The identified fragments are useful for identifying purified crotalamine. Since the peak T10 value is outside the detection range (500 to 1300 Da), it was confirmed that T10 including 2 adjacent cysteines did not appear.

Further, as shown in Fig. 9, it was confirmed that no peaks including the alkylated fragment and the free cysteine were observed under the non-reducing condition. In addition, it was confirmed that 2 desired peaks indicating correct disulfide bonds were detected between T2 and T10 (866 Da) and between T4 and T7 (1268 Da). The third disulfide bond of the purified crotamine was found to have a T6 + IAA peak at reduced mass and no peak at the non-reducing mass.

Accordingly, the crotamine protein of the present invention has three disulfide bonds such as Cys4-Cys36, Cys11-Cys30, and Cys18-Cys37 in the same manner as the previously discovered crotamine structure, and Cys4-Cys36 and Cys11-Cys30 disulfide bonds Respectively. In addition, it was confirmed that T6 participating in Cys18-Cys37 disulfide bond exerted its function under non-reducing conditions, but the presence thereof was not confirmed.

Example 6. Endotoxin assay of crotamine [

In order to test endotoxin of the final purified crotamine in Example 3, the following experiment was conducted.

More specifically, the amount of endotoxin present in the final purified crotamine was determined by quantitative Endpoint Chromogenic Limulus Amebocyte Lysate (LAL) test (Lonza, Basel, Switzerland). 50 [mu] L of purified crocamine sample or endotoxin reference samples (1.0, 0.5, 0.25 and 0.1 EU / mL) were placed in pre-warmed (37 DEG C) endotoxin-free microplate wells at 1 [mu] g / mL. Then, 50 μL of LAL reagent was added to each well and reacted at 37 ° C for 10 minutes. 100 μL of chromogenic substrate solution was added to each well, followed by incubation at 37 ° C. for 6 minutes. Then, 100 μL of quiescent reagent [25% acetic acid (v / v)] was added and analyzed on a 405 nm spectrophotometer. Endotoxin units were determined based on the standard curve obtained from the standard sample.

As a result, the endotoxin of the obtained purified crotemain was measured to be 0.15 EU / μg, which was much lower than 1 EU / μg, which is the standard required for a safe protein product, confirming that the purified crotamine was safe.

Example 7. Analysis of the effect on the expression of Kv1.3 and Kv1.5 in oocytes

In order to investigate the effect of crotamine on the hKv1.3 and hKv1.5 currents using a frog oocyte ( Xenopus oocyte) expression system, the following experiment was carried out.

7-1. Expression of Kv1.3 and Kv1.5 in oocytes

Human Kv1.3 (hKv1.3, GenBank: BC035059.1) and human Kv1.5 (hKv1.5, GenBank: BC099665.3) cRNA were transfected in vitro using mMESSAGEmMACHINE T7 kits (Ambion, Austin, TX) , And then stored at -80 ° C in distilled water without nucleic acid hydrolase (nuclease). The oocytes of step V-VI were directly isolated from the follicular and follicular membranes of female Xenopuslaevis (Nasco, Modesto, Calif.), Then cRNA was injected and the modified Barth's solution (mM) , 1 KCl, 0.4 CaCl, 0.33 Ca (NO ₃ ) ₂ , 1 MgSO ₄ , 2.4 NaHCO ₃ , 10 HEPES (pH 7.4) and 50 μg / mL gentamicin sulfate. The current was measured 3 to 6 days after injection, and the experiment was conducted according to the IACUC study guidelines of Kangwon National University.

7-2. Solution and voltage-clamp recording in oocytes

Ringer's solution (mM): 96 NaCl, 2 KCl, 1.8 CaCl ₂ , 1 MgCl ₂ and 10 HEPES (pH 7.4)) was applied to the chamber to record continuous perfusion. The solution exchange was completed within 3 minutes and the current was recorded for 15 minutes after solution exchange. The current was measured at room temperature (20-23 ° C) using a two-microelectrode voltage clamp amplifier (Warner Instruments, Hamden, CT). Electrodes were filled with 3M KCl, voltage-recording electrodes and current-passing electrodes each had 2-4 MΩ and 1-2 MΩ resistors. Stimulation and data acquisition were controlled with an AD-DA converter (Digital 1200, Axon Instruments) and pCLAMP software (v5.1, Axon Instruments). The results are shown in Fig. 10 to Fig.

As shown in Fig. 10, the hKv1.3 channel current was measured by a continuous voltage pulse from -90 mV to +60 mV per second, in 10 mV increments from every 10 seconds from a holding potential of -80 mV . It was also confirmed that crotamine reduced the Kv1.3 channel current under the condition of 3 nM crotamine treatment at +60 mV current.

Further, as shown in Fig. 11, it was confirmed that crotamine suppressed the Kv1.3 channel peak current to 40.31 + 4.72%.

In addition, as shown in FIG. 12, it was confirmed that the IC ₅₀ value according to the concentration of crotamine against the Kv1.3 channel current was 67.2 ± 44.7 nM and the Hill coefficient was 0.36 ± 0.03.

Further, as shown in Fig. 13, the hKv1.5 channel current is continuously applied from -100 mV holding potential to 10 mV for every 10 seconds, from -80 mV to +50 mV per 2 seconds, Lt; / RTI > In addition, the hKv1.5 channel current by the + 60 mV pulse in the 3 nM crotamin treated group was not changed, and it was confirmed that it was similar to the control group.

Also, as shown in Fig. 14, it was confirmed that the crotamine did not affect the Kv1.5 channel current for the pulse from -80 mV to +50 mV.

Therefore, it was confirmed that crotamine selectively suppressed Kv1.3 channel current.

<110> THE ASAN FOUNDATION <120> Recombinant microorganism producing Crotamine protein and method          for Production of Crotamine protein <130> asan1-105 <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> crotamine protein <400> 1 Tyr Lys Gln Cys His Lys Lys Gly Gly His Cys Phe Pro Lys Glu Lys   1 5 10 15 Ile Cys Leu Pro Pro Ser Ser Asp Phe Gly Lys Met Asp Cys Arg Trp              20 25 30 Arg Trp Lys Cys Cys Lys Lys Gly Ser Gly          35 40 <210> 2 <211> 378 <212> PRT <213> Artificial Sequence <220> MBP (maltose-binding protein) <400> 2 Met Gly Ser Ser His His His His His Gly Thr Lys Thr Glu Glu   1 5 10 15 Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu              20 25 30 Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr          35 40 45 Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala      50 55 60 Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly  65 70 75 80 Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala                  85 90 95 Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn             100 105 110 Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile         115 120 125 Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile     130 135 140 Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met 145 150 155 160 Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp                 165 170 175 Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp             180 185 190 Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val         195 200 205 Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile     210 215 220 Ala Glu Ala Ala Phe Asn Lys Gly Glu Glu Thr Ala Met Thr Ile Asn Gly 225 230 235 240 Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val                 245 250 255 Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly             260 265 270 Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala         275 280 285 Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala     290 295 300 Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu 305 310 315 320 Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala                 325 330 335 Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp             340 345 350 Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr         355 360 365 Val Asp Glu Ala Leu Lys Asp Ala Gln Thr     370 375 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> His6 (hexahistidine) protein <400> 3 Met Gly Ser Ser His His His His His His   1 5 10 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Trx (thioredoxin) protein <400> 4 Met Gly Ser Ser His His His His His Gly Thr Ser Asp Lys Ile   1 5 10 15 Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp              20 25 30 Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys          35 40 45 Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys      50 55 60 Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro  65 70 75 80 Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly                  85 90 95 Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys             100 105 110 Glu Phe Leu Asp Ala Asn Leu Ala         115 120 <210> 5 <211> 231 <212> PRT <213> Artificial Sequence <220> GST (glutathione S-transferase) <400> 5 Met Gly Ser Ser His His His His His Gly Thr Ser Pro Ile Leu   1 5 10 15 Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu              20 25 30 Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu          35 40 45 Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro      50 55 60 Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met  65 70 75 80 Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys                  85 90 95 Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp             100 105 110 Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr         115 120 125 Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe     130 135 140 Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr 145 150 155 160 His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met                 165 170 175 Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys             180 185 190 Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys         195 200 205 Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly     210 215 220 Asp His Pro Pro Lys Ser Asp 225 230 <210> 6 <211> 279 <212> PRT <213> Artificial Sequence <220> PDIb'a '(protein disulfide isomerase b'a' domain) <400> 6 Met Lys His His His His His His His Glu Leu Ile Glu Phe Thr   1 5 10 15 Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His Ile              20 25 30 Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys Leu Ser          35 40 45 Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu Phe Ile      50 55 60 Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg Ile Leu Glu Phe Phe  65 70 75 80 Gly Leu Lys Lys Glu Glu Cys Pro Ala Val Arg Leu Ile Thr Leu Glu                  85 90 95 Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala Glu             100 105 110 Arg Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys Pro         115 120 125 His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro Val     130 135 140 Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu Lys 145 150 155 160 Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys                 165 170 175 Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp His             180 185 190 Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val Glu         195 200 205 Ala Val Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala Ser     210 215 220 Ala Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp Gly 225 230 235 240 Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp Asp                 245 250 255 Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu Glu             260 265 270 Asp Asp Asp Gln Lys Ala Val         275 <210> 7 <211> 499 <212> PRT <213> Artificial Sequence <220> PDI (protein disulfide bond isomerase) <400> 7 Met Lys His His His His His His His Glu Leu Asp Ala Pro Glu   1 5 10 15 Glu Glu Asp His Val Leu Val Leu Arg Lys Ser Asn Phe Ala Glu Ala              20 25 30 Leu Ala Ala His Lys Tyr Leu Leu Val Glu Phe Tyr Ala Pro Trp Cys          35 40 45 Gly His Cys Lys Ala Leu Ala Pro Glu Tyr Ala Lys Ala Ala Gly Lys      50 55 60 Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala Lys Val Asp Ala Thr  65 70 75 80 Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val Arg Gly Tyr Pro Thr                  85 90 95 Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser Pro Lys Glu Tyr Thr             100 105 110 Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp Leu Lys Lys Arg Thr         115 120 125 Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly Ala Ala Ala Glu Ser Leu     130 135 140 Val Glu Ser Ser Glu Val Ala Val Ile Gly Phe Phe Lys Asp Val Glu 145 150 155 160 Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala Ala Glu Ala Ile Asp Asp                 165 170 175 Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp Val Phe Ser Lys Tyr Gln             180 185 190 Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys Phe Asp Glu Gly Arg         195 200 205 Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn Leu Leu Asp Phe Ile     210 215 220 Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe Thr Glu Gln Thr Ala 225 230 235 240 Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His Ile Leu Leu Phe Leu                 245 250 255 Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys Leu Ser Asn Phe Lys Thr             260 265 270 Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu Phe Ile Phe Ile Asp Ser         275 280 285 Asp His Thr Asp Asn Gln Arg Ile Leu Glu Phe Phe Gly Leu Lys Lys     290 295 300 Glu Glu Cys Pro Ala Val Arg Leu Ile Thr Leu Glu Glu Glu Met Thr 305 310 315 320 Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala Glu Arg Ile Thr Glu                 325 330 335 Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys Pro His Leu Met Ser             340 345 350 Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro Val Lys Val Leu Val         355 360 365 Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu Lys Lys Asn Val Phe     370 375 380 Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Gln Leu Ala Pro 385 390 395 400 Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp His Glu Asn Ile Val                 405 410 415 Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val Glu Ala Val Lys Val             420 425 430 His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala Ser Ala Asp Arg Thr         435 440 445 Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp Gly Phe Lys Lys Phe     450 455 460 Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp Asp Asp Asp Leu Glu 465 470 475 480 Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu Glu Asp Asp Asp Gln                 485 490 495 Lys Ala Val             <210> 8 <211> 506 <212> PRT <213> Artificial Sequence <220> NuA (N-utilizing substance protein A) <400> 8 Met Gly Ser Ser His His His His His Gly Thr Asn Lys Glu Ile   1 5 10 15 Leu Ala Val Val Glu Ala Val Ser Asn Glu Lys Ala Leu Pro Arg Glu              20 25 30 Lys Ile Phe Glu Ala Leu Glu Ser Ala Leu Ala Thr Ala Thr Lys Lys          35 40 45 Lys Tyr Glu Gln Glu Ile Asp Val Arg Val Gln Ile Asp Arg Lys Ser      50 55 60 Gly Asp Phe Asp Thr Phe Arg Arg Trp Leu Val Val Asp Glu Val Thr  65 70 75 80 Gln Pro Thr Lys Glu Ile Thr Leu Glu Ala Ala Arg Tyr Glu Asp Glu                  85 90 95 Ser Leu Asn Leu Gly Asp Tyr Val Glu Asp Gln Ile Glu Ser Val Thr             100 105 110 Phe Asp Arg Ile Thr Thr Gln Thr Ala Lys Gln Val Ile Val Gln Lys         115 120 125 Val Arg Glu Ala Glu Arg Ala Met Val Val Asp Gln Phe Arg Glu His     130 135 140 Glu Gly Glu Ile Ile Thr Gly Val Val Lys Lys Val Asn Arg Asp Asn 145 150 155 160 Ile Ser Leu Asp Leu Gly Asn Asn Ala Glu Ala Val Ile Leu Arg Glu                 165 170 175 Asp Met Leu Pro Arg Glu Asn Phe Arg Pro Gly Asp Arg Val Gly             180 185 190 Val Leu Tyr Ser Val Arg Pro Glu Ala Arg Gly Ala Gln Leu Phe Val         195 200 205 Thr Arg Ser Lys Pro Glu Met Leu Ile Glu Leu Phe Arg Ile Glu Val     210 215 220 Pro Glu Ile Gly Glu Glu Glu Ile Glu Ile Lys Ala Ala Ala Arg Asp 225 230 235 240 Pro Gly Ser Arg Ala Lys Ile Ala Val Lys Thr Asn Asp Lys Arg Ile                 245 250 255 Asp Pro Val Gly Ala Cys Val Gly Met Arg Gly Ala Arg Val Gln Ala             260 265 270 Val Ser Thr Glu Leu Gly Gly Glu Glu Arg Ile Asp Ile Val Leu Trp Asp         275 280 285 Asp Asn Pro Ala Gln Phe Val Ile Asn Ala Met Ala Pro Ala Asp Val     290 295 300 Ala Ser Ile Val Val Asp Glu Asp Lys His Thr Met Asp Ile Ala Val 305 310 315 320 Glu Ala Gly Asn Leu Ala Gln Ala Ile Gly Arg Asn Gly Gln Asn Val                 325 330 335 Arg Leu Ala Ser Gln Leu Ser Gly Trp Glu Leu Asn Val Met Thr Val             340 345 350 Asp Asp Leu Gln Ala Lys His Gln Ala Glu Ala His Ala Ala Ile Asp         355 360 365 Thr Phe Thr Lys Tyr Leu Asp Ile Asp Glu Asp Phe Ala Thr Val Leu     370 375 380 Val Glu Glu Gly Phe Ser Thr Leu Glu Glu Leu Ala Tyr Val Pro Met 385 390 395 400 Lys Glu Leu Leu Glu Ile Glu Gly Leu Asp Glu Pro Thr Val Glu Ala                 405 410 415 Leu Arg Glu Arg Ala Lys Asn Ala Leu Ala Thr Ile Ala Gln Ala Gln             420 425 430 Glu Glu Ser Leu Gly Asp Asn Lys Pro Ala Asp Asp Leu Leu Asn Leu         435 440 445 Glu Gly Val Asp Arg Asp Leu Ala Phe Lys Leu Ala Ala Arg Gly Val     450 455 460 Cys Thr Leu Glu Asp Leu Ala Glu Gln Gly Ile Asp Asp Leu Ala Asp 465 470 475 480 Ile Glu Gly Leu Thr Asp Glu Lys Ala Gly Ala Leu Ile Met Ala Ala                 485 490 495 Arg Asn Ile Cys Trp Phe Gly Asp Glu Ala             500 505 <210> 9 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> purified crotamine <400> 9 Gly Tyr Lys Gln Cys His Lys Lys Gly Gly His Cys Phe Pro Lys Glu   1 5 10 15 Lys Ile Cys Leu Pro Pro Ser Ser Asp Phe Gly Lys Met Asp Cys Arg              20 25 30 Trp Arg Trp Lys Cys Cys Lys Lys Gly Ser Gly          35 40 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> attB1 primer <400> 10 ggggacaagt ttgtacaaaa aagcaggctt c 31 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB2 primer <400> 11 acccagcttt cttgtacaaa gtggtcccc 29

Claims (18)

DNA encoding His6 (hexahistidine) protein; A protein disulfide isomerase (PDI), a protein disulfide isomerase (PDI) domain, a protein disulfide isomerase (PDI) domain, and a N-abiotic substance a DNA encoding at least one tag protein selected from the group consisting of a protein A); DNA encoding the TEV (Tavacco Etch Virus) protease recognition site; And a DNA encoding the amino acid sequence of crotamine represented by SEQ ID NO: 1. delete The method according to claim 1,
2. The recombinant vector for producing a crotamine protein according to claim 1, wherein the MBP is an amino acid sequence represented by SEQ ID NO: 2. delete The method according to claim 1,
Wherein the Trx comprises an amino acid sequence represented by SEQ ID NO: 4. The method according to claim 1,
Wherein said GST comprises an amino acid sequence represented by SEQ ID NO: 5. The method according to claim 1,
Wherein said PDIb'a 'is an amino acid sequence represented by SEQ ID NO: 6. The method according to claim 1,
Wherein the PDI comprises the amino acid sequence shown in SEQ ID NO: 7. The method according to claim 1,
8. The recombinant vector for producing a crotamine protein according to claim 7, wherein said NusA is an amino acid sequence represented by SEQ ID NO: 8. delete delete delete The method according to claim 1,
Wherein the recombinant vector has a cleavage map as set forth in the following figure.
[drawing]

9. A recombinant microorganism producing a crotamine protein transformed with the recombinant vector of any one of claims 1, 3, 5 to 9. 15. The method of claim 14,
Wherein the recombinant microorganism is at least one member selected from the group consisting of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast. 1) culturing the recombinant microorganism of claim 14 and obtaining a culture; And
2) separating and purifying the chromatin protein from the culture solution. 17. The method of claim 16,
Wherein the recombinant microorganism is cultured at 15 to 40 DEG C in the step 1). 17. The method of claim 16,
Wherein the crotamine produced by the production method of the crotamine protein is an amino acid sequence of SEQ ID NO: 9.

Images