KR100384467B1 - Lumburokinase gene isolated from a earthworm and overexpression method of lumburokinase protein in yeast - Google Patents

Abstract

The present invention reveals a novel rumburokinase III-2 coding gene sequence and amino acid sequence in a Korean earthworm (Lumbricus rubellus) and mass expression of rumburokinase using Pichia pastoris, a yeast expression system. It is about a method. Using the yeast expression system of the present invention can not only obtain a large amount of recombinant rumbaurokinase protein, but also obtain high purity by applying a simple separation and purification process. Recombinant rumburokinase protein expressed in yeast through the present invention has a high fibrinolytic activity and high medical value.

Description

Lumburokinase gene isolated from a earthworm and overexpression method of lumburokinase protein in yeast}

The present invention relates to a method for mass expression of a lumpurokinase gene isolated from an earthworm and a yeast, and more particularly, an amino acid sequence of a lumpurokinase protein having a thrombolytic ability isolated from a worm from Korea. Nucleotide sequence of the coding gene, and expression vector containing the rumbaurokinase coding gene and Pichia pastoris, a transformed recombinant yeast host expressing rumbaurokinase.

Patents on the method of expressing and purifying a useful protein of interest using genetic recombination technology are increasing worldwide. In particular, mass expression of proteins using yeast as an expression bath makes the useful protein easy to be purified outside the cell by fusion of secretion signal sequence and the protein of interest. Pichia pastoris has a very high growth rate, which can produce large amounts of protein per liter of culture and produce very low levels of protein. Instead, secrete foreign proteins up to grams per liter in cheap protein-poor medium. It has a big advantage. In addition, transcription of genes transduced into Pichia pastoris is tightly regulated by the AOX1 (alcohol oxidase gene) promoter and rapid transcription is induced upon addition of methol. Therefore, studies are currently underway to produce a variety of proteins using the expression host Pichia pastoris, and already transformed Pichia pastoris producing foreign proteins are disclosed in US Pat. Nos. 4,683,293, 4,808,537, and 4,812,405. US Patent No. 4,818,700, US Patent 4,837,148, US Patent 4,855,231, US Patent 4,857,467, US Patent 4,879,231, US Patent 4,882, 279, US Patent 4,885,242, US Patent 4,895,800 US Patent No. 4,929,555, US Patent 5,002,876, US Patent 5,004,688, US Patent 5,032,516, US Patent 5,122,465, US Patent 5,135,868, US Patent 5,166,329, and the like.

One of the adult diseases that increase as living standards and diets gradually become westernized is bloodstream disease. In particular, myocardial infarction, stroke, and venous thrombosis are caused by the formation of abnormal blood clots in the blood-related diseases, which are very rapidly increasing in the modern society, leading to great social conviction. The hemostatic system smoothly moves blood to blood vessels and repairs damaged blood vessels to prevent blood loss when blood vessels are damaged by external stimuli (Colman, RW, Hirsh, J.). , Marder, VJ, and Salzman, EW, 1994. Lippincott company, USA) The hemostatic system consists largely of blood vessels, platelets, and soluble plasma proteins. Platelets act as a temporary protection against damage to blood vessels, and plasma proteins and chemicals are used to reinforce the hemostatic plug. These plasma proteins are involved in blood coagulation as well as fibrinolysis. Despite the role of the hemostatic system, blood clots occur in the body when thrombogenic stimulation takes place externally. The main thrombogenic factors are blood cells, fibrin and platelets, which are the main components of blood clots formed by vascular endothelial cells falling off and exposure to the blood vessels. However, the composition changes depending on the production environment. Venous thrombosis consists of red blood cells, large amounts of fibrin, and small amounts of platelets. Arterial thrombosis, on the other hand, is mainly due to platelet aggregation and relatively small amounts of fibrin are present. Since these clots actually cause serious diseases, we are currently exploring and studying thrombolysis from various sources for the purpose of treatment and prevention.

The thrombolytic agents currently used clinically include haparin (Gruber, UF, Saldeen, T., and Brokop, T., 1980, Br Med J, 1, 69), urokinase (White, WF, Borlow, GH, and Mozen, MM, 1966. Biochem, 5, 2160), streptokinase (Davies, MC, Englert, ME, and De Renzo, EC 1964. J Biol Chem, 239 (8), 2651-2656), histoplasmoplasmin Tissue-type plasminogen activators (Brott, TG, Haley, EC, and Levy, DE, 1992, Stroke, 23.632) have been consumed in large quantities worldwide. However, these thrombolytic agents do not directly dissolve fibrin, a major component of thrombus, but activate plasminogen that is already present in the blood with plasmin, thereby allowing plasmin to dissolve the thrombus. Therefore, the use of these thrombolytic agents can often cause systemic bleeding by causing excessive plasmon formation and dissolving to the blood vessel walls, which can be life threatening.

In order to solve this problem, other proteins in blood vessels do not attack relatively well, but the search and development of thrombin specific solubilizers with direct fibrinolytic ability are underway by many researchers from various sources. Representative thrombus specific solubilizers currently known in this effort are fibrinase isolated from snake venom, which are fibrolase (Retzios, AD, and Markland, FS 1994. Thromb Res, 74 (4), 355-367 Ahmed, NK, Tennant, KD, Markland, FS, and Lacz, JP 1990. Haemostasis, 20 (3), 147-154, basiliscus fibrases: Retzios, AD, and Markland, F.Jr. 1992. Biochem, 31 (19), 4547-4557), ARHs (Lollar, P., Parker, CG, Kajenski, PJ, Litwiller, RD, and Fass, DN 1987. Biochem, 26 (24), 7627-7636. ), And atroxase (Tu, AT, Baker, B., Wongvibulsin, S., Willis, T. 1996. Toxicon, 34 (11), 1295-1300), lebetase (Siigur, E., Aaspollu, A., Tu, AT, and Siigur, J. 1996. Biochem Biophys Res Commun, 224 (1), 229-236). In addition, in Korea, 28.2 kDa protease was extracted from CK 11-4 in Bacillus genus Cheonggukjang (Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I., and Lee, S. 1996. Appl Environ Microbiol, 62 (7), 2482-2488), Plasminogen Ratio from Korean Tabanus fulvus Two proteins (22 kDa and 27 kDa) with dependent fibrinolytic activity were isolated.

Earthworms have been widely used in folk medicine for vascular diseases in the East, including China, Korea, and Japan. Recently, six types of proteins that display thrombolytic activity from earthworms have been isolated and named lumbrokinase (LK) (Mihara, H., Sumi, H., Yoneta, T., Mizumoto, H., Ikeda, R., Seiki, M., and Maruyama, M. 1991. Jap J Physiol, 41, 461-472), these proteins can dissolve fibrin even in the absence of plasminogen. They are stable to temperature and pH changes, act selectively on fibrin directly without activating plasminogen as plasmin, and do not degrade proteins such as albumin or casein that are not involved in thrombus formation. Because of this, it is very suitable for therapeutic thrombolysis. Lumburokinase is currently used as a surface treatment agent for preventing thrombus formation when artificial kidneys are made of urethane. (2) Powder preparations made by drying the supernatant after centrifugation by grinding earthworms are used as oral thrombolytic agents. Table 1 below shows the simple biochemical and physical properties of rumbaurokinases.

TABLE 1 Lumburokinase I II III 0 One 2 One 2 Molecular weight (kDa: analyzed by electrophoresis) by isoelectric focusing E ^1% (280 nm) 23.54.1212.5 27.44.0014.2 27.03.9012.8 28.53.8016.6 34.03.7013.1 34.23.5212.7 Classification Chymotrypsin system Does not belong to chymotrypsin or trypsin system Trypsin system Thermal stability 37 ° C, pH 2-10: Has activity of 80% or more; 60 ° C, pH 6-9: Has activity of 60% or more

The method for purifying rumbaurokinase is known from Mihara et al in Japan (Japanese Patent Laid-Open No. 58-148824, Japanese Patent Laid-Open No. 59-63184, Japanese Patent Laid-Open No. 59-184131, Japanese Patent Laid-Open No. 64). -75431), a new method for separating and purifying lumpurokinase was also developed in Korea. (Domestic Patent No. 92-8369) Recently, a column of diethylaminoethyl-Sephadex anion exchange (DEAE-Sephadex anion exchange) A method for the easy separation of rumbaurokinase from earthworms using only p-amino benzamidine affinity column chromatography has been reported, and the cleavage pattern of fibrinogen by rumbaurokinase and the cleavage of cleavage sites on its substrate It has also been identified that rumbaurokinase hydrolyzes immediately after lysine (133th and 148th of the Bβ-chain) (Park, YD, Kim, JW, Min, BG, Seo, JW, and Jeong, JM 1998. Biotech Let, 20 (2), 169-172) However, it is not only time-consuming and expensive to separate and purify rumburokinase from the earthworm, but also pure separation of one kind of lumpurokinase. At the same time, at least five different proteases in the earthworm body are obtained at the same time, so complex separation and purification steps are not suitable for mass production processes.

Therefore, the method of purifying and isolating lumpurokinase directly from the earthworm is not only easy to obtain a single type of pure and expensive lumpurokinase due to the cost and time, but also when the source worm is contaminated with heavy metal, It is a problem.

In order to overcome the above-mentioned problems, the present invention provides a recombinant vector for producing a large amount of rumbaurokinase having a thrombolytic ability of a natural type, to a high yield of Rumbu through precise induction control in transformed host cells. It aims at manufacturing a kinase.

Accordingly, an object of the present invention is to provide an amino acid sequence and a gene sequence encoding the new rumburokinase protein in the earthworm.

It is another object of the present invention to provide a recombinant vector containing a gene encoding the rumbukinase and capable of producing a recombinant rumburokinase protein.

In addition, an object of the present invention is to provide a large amount of recombinant rumbaurokinase having thrombolytic ability using yeast as an expression host.

FIG. 1 compares and analyzes the amino acid sequences of the Lumburokinase III-2 amino acid sequence of the present invention and the previously known Lumburokinase III-1 and Lumburokinase 1T4.

2 is a comparison of the rumburokinase 1T4 coding gene, which has already been cloned from the earthworm and whose gene sequencing has been identified, and the nucleotide sequences of the rumburokinase III-2 coding gene of the present invention.

Figure 3 is a schematic of the introduction of the rumbaurokinase III-2 coding gene into the yeast expression vector pPIC9K of the present invention,

Figure 4 is a recombinant rumburokinase III-2 gene in the present invention was expressed on Pichia pastoris (Pichia pastoris) and confirmed on SDS-PAGE,

FIG. 5 shows the enzymatic activity of recombinant rumbaurokinase III-2 of the present invention on fibrin plate media.

In order to achieve the above object, the present invention is a Lumbricus kinase III-2 protein having a novel thrombolytic ability from the Korean earthworm ( Lumbricus rubellus ); A gene base sequence of SEQ ID NO: 1 encoding the above; Primer 1 and primer 2 used for PCR of the gene; And Roomburokinase III-2 protein comprising the amino acid sequence of SEQ ID NO: 2 deduced from SEQ ID NO: 1.

In another aspect, the present invention provides a recombinant vector into which the Lumburokinase III-2 coding gene is inserted.

The present invention also provides a recombinant host cell expressing the recombinant vector.

In another aspect, the present invention provides a method for inducing the expression of the rumbaurokinase III-2 protein with methanol after culturing the transformed yeast.

Hereinafter, the present invention will be described in detail.

The present inventors crushed the earthworm to separate the thrombolytic proteins present in the earthworm, and then filtered by anion exchange column chromatography and affinity column chromatography to separate the fraction with high fibrinolytic activity. Thereafter, the amino acid sequence of the isolated protein (SEQ ID NO: 2) was determined and injected into mice to obtain anti-lumburokinase serum.

In addition, cDNA libraries were prepared by isolating the mRNA of the earthworm, and clones containing the cDNA codec of lumpurokinase were selected using anti-lumburokinase serum and cloned. The obtained clones were analyzed by restriction enzyme maps, and among them, clones having the same restriction enzyme map as rumbaurokinase-1T4 were selected to determine gene sequences.

The cloned rumbaurokinase coding gene of SEQ ID NO: 1 was termed rumbaurokinase III-2 and inserted into the pPIC9K vector, a yeast expression vector, to prepare a recombinant pPIC9K / LK III-2 vector.

The pPIC9K vector used in the present invention contains an alpha-factor leader sequence of Saccharomyces cerevisiae that secretes the expressed protein out of the yeast, and His4 (histidinol dehydrogenase) gene. , Ampicillin and kanamicin genes are included, so it is easy to check whether or not the transduction into the host.

BMMY (1% Bactobacillus yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0), a protein-poor medium for expression of rumbaurokinase in recombinant host cell Pichia pastoris KFCC-11166 of the present invention , 1.24% yeast nitrate, 4 × 10 ⁻⁵ % biotin, 0.5% methanol) was used and expression was induced for 4 days at 30 ° C. at 240 rpm with methanol added to the medium every 0.5 hours. Rumburokinase secreted into the culture medium had a difference in expression depending on the culture conditions, but after 96 hours of expression induction using methanol in aerobic conditions by the flask culture method, 5 mg of recombinant lumpurokinase per liter was obtained. Recombinant Lumburokinase has the same thrombolytic activity as that of natural Lumburokinase and has a yield three times higher than that of Lumburokinase extracted from the existing earthworm.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples illustrate the present invention in detail, and the scope of the present invention is not limited by the following examples.

Example 1

Pure Separation and Amino Acid Sequence Determination of Lumburokinase from Earthworms

(1) Separation and Purification of Lumburokinase from Earthworms

① Acquisition of earthworm extract

3 kg of live earthworms were washed with tap water and placed in four volumes of TBS (10 mM Tris, 150 mM sodium chloride, pH 7.4) and ground with a blender. After stirring for 4 days at room temperature, the supernatant was taken by centrifugation at 12,000 g for 30 minutes at 4 ℃.

② heat treatment

The supernatant of ① was heat-treated at 60 ° C. for 1 hour and centrifuged at 12,000 g for 30 minutes at 4 ° C. The supernatant was taken and 0-30% (w / v) of ammonium sulfate was added slowly at 0 ° C., followed by centrifugation. The precipitate was suspended and stored in 50 mM Tris buffer (pH 8.0). The supernatant was again slowly added 30-60% (w / v) ammonium sulfate at 0 ° C. and then centrifuged to discard the supernatant and the precipitate was combined with the precipitate formed first. This precipitate suspension was dialyzed with 50 mM Tris pH 8.0 buffer.

③ Perform anion exchange column

The dialysed protein mixture was injected into a diethylaminoethyl Sephadex A25 column (DEAE-Sephadex A25 column; 5 cm x 100 cm; width x length) and washed with 20 mM Tris buffer (pH 8.0). Receive fractions of 8 ml by flowing 3 ml per minute in a linear gradient of 0-500 mM sodium chloride to the column. 10 μl of the fraction was dropped onto fibrin plate media (Astrup, T., and Mullertz, S., 1952. Arch Biochem Biophys, 40, 346-351). Then, the plate medium was reacted for 2 hours at 37 ℃, the fibrinolytic activity was measured.

④ Affinity column

The fractions of the high fibrin activity of ③ are collected and injected into a ρ-aminobenzamidine Sepharose 6B column (2.5 cm x 10 cm; width x length), followed by 20 mM Tris buffer solution. washed with pH 8.0. Elution buffer (500 mM alginine, 20 mM sodium acetate, pH 5.0) was flowed in 1 ml at a minute while receiving 1 ml fractions, and 5 µl of the fraction was dropped onto the fibrous plate medium. Afterwards, the fibrinolytic activity was measured for 1 hour at 37 ° C. Based on the results, the fractions showing high enzyme titer were collected and dialyzed with 50 mM Tris buffer solution (pH 8.0) and centrifrep (Amicon, USA). Concentration gave rumburokinase.

(2) amino acid sequence analysis

After separation and denaturation of the separated lumburokinase by SDS-PAGE and staining with Coomassie brilliant blue to confirm the position of the rumburokinase, cut only that part from the gel electro-eluter (Bio-eluter: Bio The protein was eluted with -Rad). A small amount is added every 0 min, 10 min, 30 min, 2 h, 6 h, 18 h while reacting at 37 ° C with the addition of active rumbu kinase or endoproteinase Lys-C. Take 0.5 volume fold of SDS-PAGE sample buffer (50 mM Tris buffer, 5% (w / v) SDS, 200 mM DTT, 5 mM EDTA, 20% (v / v) glycerol, and a small amount of pyronin Y ) Was stopped by the addition of). Samples taken each time were analyzed by SDS-PAGE, and the reaction time was selected in the same manner by selecting the most responsive time among the cut peptides, followed by SDS-PAGE. Fractionated peptides were transferred from gels to polyvinidene diflu in electroblot buffer (10 mM 3- [cyclohexylamino] -1-propanesulphonic acid (CAPS), pH 11, 10% (v / v) methanol). The lead (PVDF: polyvinyidene difluoride) membrane was moved to 300 mA for 90 minutes and stained to determine the position of the band. The amino acid sequence analysis of each protein band stained was commissioned by the Korea Institute of Basic Science.

In Fig. 1, the sequences of the novel Lumburokinase III-2 and the previously known amino acids of Lumburokinase III-1 and Lumburokinase 1T4 are compared. Lumburokinase III-2 showed a large difference from Lumburokinase III-1, but only 4 amino acids which were reversed from Lumburokinase 1T4 were inconsistent and all were identical.

Example 2

Earthworm cDNA Library Preparation and Lumburokinase Coded cDNA Screening (1) Earthworm cDNA Library Preparation

① Extraction of whole RNA from earthworm

The live earthworms were washed with tap water, and only 24 hours of water was used to excrete the wastes in the intestine. The wastes were removed with distilled water and the earthworms were frozen in liquid nitrogen and stored at -80 ℃. After between the worm frozen Muller placed in the bowl intermittent addition of liquid nitrogen and finely until the powder, and into the 100 ㎍ earthworm powder in the tube was added a 1 ㎖ ultra specification II RNA reagent (Ultraspec II RNA Reagent ^ⓡ) Completely dissolved. After reacting for 5 minutes on ice, 0.2 ml of chloroform was added and mixed thoroughly, followed by reaction for 5 minutes on ice. The supernatant was recovered by centrifugation at 12,000 g for 15 minutes at 4 ° C. and mixed well by adding 0.5 volume of isopropanol and 0.05 volume of Ultraspec II RNA reagent to the supernatant. The mixture was centrifuged to remove the supernatant, washed twice with 70% ethanol (pellets) with 70% ethanol, and dried. The RNA pellet was dissolved in 50 μl of distilled water treated with DEPC (diethyl pyrocarbonate), and then centrifuged again to obtain only the supernatant. An RNA sample was prepared by adding the same amount of 2 × column injection buffer (40 mM Tris, 1 M lithium chloride, 2 mM EDTA, 0.2% (w / v) SDS, pH 7.6).

② Oligo dT column preparation for mRNA separation

0.1 g of oligo dT cellulose powder was added to 5 ml of 0.1 N sodium hydroxide and activated at 4 ° C. for 12 hours. Oligo dT cellulose was fixed in the finished pipette pipette with glass wool, followed by distilled water mixed with 1 ml of 0.1 N sodium hydroxide, 5 ml of diethyl pyrocarbonat, and 5 ml of 1 × column injection buffer. Wash and equilibrate.

③ mRNA isolation

The RNA sample of ① was injected into the column of ② and washed with 10 ml of 1 × column injection buffer. In addition, the RNA sample was eluted with 3 ml of elution buffer (10 mM Tris, 1 mM EDTA, 0.05% SDS, pH 7.6) and the absorbance was measured at 260 nm to collect the fraction containing mRNA. Fractions were extracted with phenol, precipitated with ethanol and finally concentrated to 45 μl of solution to obtain mRNA.

④ cDNA synthesis

The primary strand cDNA and the secondary strand cDNA were synthesized by the following method. 15 μl of the mRNA solution of ③ was heated at 80 ° C. for 5 minutes, and then left on ice for 5 minutes. The oligo dT primers, dNTPs, RNase inhibitors and murine reverse transcriptase were added thereto, and then reacted at 37 ° C. for 2 hours to synthesize primary cDNA strands containing mRNA. RNase H and E. coli DNA polymerase I, [α-32P] dCTP were added to the synthesized primary cDNA reactant at 16 ° C. for 5 hours, followed by addition of E. coli DNA conjugated enzyme and T4 bacteriophage polynucleotide kinase. After minute reaction, EDTA (pH 8.0) was added to finally synthesize double stranded cDNA. Double stranded cDNA was extracted with phenol, precipitated with ethanol and suspended in 60 μl of TE buffer.

For repair and methylation of double-stranded cDNA, S-adenosyl-L-methionine and EcoRI-methylase were added to 60 μl of double-stranded cDNA and reacted at 37 ° C. for 1 hour. Heat treated at 68 ° C. for 15 minutes. After phenol extraction, it was precipitated with ethanol and suspended in final 29 μl of TE buffer. Then, the suspension was heat-treated at 68 ° C. for 5 minutes, cooled to 37 ° C., and then dNTPs and bacteriophage T4 polymerase were added and reacted again at 37 ° C. for 30 minutes. The reaction was then stopped with EDTA (pH 8.0), extracted with phenol and precipitated with ethanol to resuspend cDNA with 10 mM Tris buffer (pH 7.6).

⑤ Lumbrokinase cDNA EcoR I linker junction

EcoR I linker was conjugated to cDNA of ④. The following reaction mixture was mixed with 13 μl of cDNA solution for 12 hours at 16 ° C. 2 μl of 10 × conjugase buffer 2 μl of 500 μng / μl EcoR I linker 1 μl of 1 U / μl T4 DNA conjugate 2 μl 10 mM ATP

0.5 μl of the reaction was transferred to a new tube, and the remaining 19.5 μl was heat treated at 68 ° C. for 15 minutes to stop the reaction, and then left on ice for 5 minutes. Then, the following solutions were added and reacted at 37 ° C. for 2 hours. 20 μl of 10 × EcoR I buffer 2 μl of 12 U / μl EcoR I 150 μl of sterile water

After the reaction, the phenol was extracted, precipitated with ethanol and suspended in the final 20 μl of 10 mM Tris buffer (pH 7.6). ⑥ cDNA conjugation and packaging to the lambda vector 2 μl of 20 μl of the reaction was taken to The mixture was added thereto, reacted for 12 hours at 16 ° C., and then stored at 4 ° C. 4 μl of 250 ng / μl λgt 11 / EcoR I arms 1 μl of 10 × conjugate enzyme buffer 1 μl of 10 mM ATP1 μl of 1 U 1 μl of sterile water

Gigapack II Gold-7 Packaging Extract Kit (Stratagene, USA) was used to transfer the reaction to bacteriophages. Take out freeze / thaw extract at -80 ° C and quickly dissolve. Add 4 μl of cDNA, leave on ice, dissolve Sonic Extract and take 15 μl of freeze / saw with cDNA. The mixture was added to the extract tube, mixed well, and reacted at room temperature for 2 hours. To this was added 500 μl SM buffer (50 mM Tris, 100 mM sodium chloride, 8 mM magnesium sulfide, 0.1% (w / v) gelatin, pH 7.5) and 20 μl of chloroform and centrifuged for 30 seconds, The supernatant was taken and stored at 4 ° C.

⑦ titration and amplification of cDNA library

200 μl of E. coli (Y1090, A600 = 0.5) was added to six culture tubes, and 20 μl of a solution diluted 1/10 to 1/10 ⁶ of the cDNA library was added to each tube, followed by incubation at 37 ° C. for 15 minutes. . 5 mL NZY upper layer agar (1% (w / v)) NZ amine (casein hydrolysate), 0.5% yeast extract, 0.5% (w / v) sodium chloride, 0.2% (w / v) magnesium hydride sulfide, 0.7 % (w / v) agar) was added to the tube, mixed to achieve final concentrations of IPTG and X-gal of 1.5 mM and 1.2 mM, respectively, and then plated onto the underlying 1.5% (w / v) NZY agar plate. . After stiffening the upper agar medium, it was incubated for 8 hours at 37 ℃ and titrated plaque forming unit (pfu) and based on this result 2 × 10 ⁴ pfu / 20 μl and 200 μl of E. coli culture (A ₆₀₀ = 0.5 ) Was mixed in a culture tube and incubated at 37 ° C. for 15 minutes, and then 5 ml of NZY upper layer agar medium was added to NZY lower layer agar plate. After 4 hours of incubation at 37 ° C., when plaque appeared, 3 ml of SM buffer was poured into the plate medium and reacted at 4 ° C. for 12 hours to mix the bacteriophage with SM buffer. SM buffer solution mixed with bacterial phage was collected, 50 μl of chloroform was added, and the supernatant was stored at 4 ° C. by centrifugation for 30 seconds.

(2) Lumburokinase coding gene selection from cDNA library

An anti-lumburokinase sera capable of probing the roomburokinase protein was selected to select the lumburokinase coding gene from the cDNA library.

① Serum Production of Anti-Lumburokinase

In Example 1, the purified Lumburokinase from the earthworm was separated on SDS-PAGE, and 34 kDa protein bands corresponding to Lumburokinase were removed from the gel, and then pulverized to obtain the same amount of Freund's adjuvant. Mixed together.

1st injection: Complete adjuvant

2nd-4th injections: Incomplete adjuvant

The mixture was pulverized as fine as possible by passing the mixture five times with a small-bore syringe, and 50 μg of rumbaurokinase protein was injected per experimental white paper (ICR strain).

Second injection: 15 days after first injection

3rd injection: 15 days after 2nd injection

4 th injection: 10 days after 3 th injection

Five days after the fourth injection, the blood collected by cardiac collection was left at room temperature for 10 minutes and then centrifuged at 12,000 g for 15 minutes, and the supernatant was transferred to a new tube and stored at -80 ° C.

② Lumbrokinase coding gene selection

Iii) primary screening

The cDNA library was infected with E. coli and cultured at 37 ° C. so that 2 × 10 ⁴ plaques appeared per agar plate medium. When plaques appeared to be 1 mm in diameter, a nitrocellulose membrane treated with 10 mM IPTG (isopropyl-β-D-thiogalactopyranoside) solution was covered on the medium and incubated at 37 ° C for 12 hours. Place the tip of the pipette tip on the membrane and agar medium, wash the membrane with blocking buffer (10 mM Tris, 150 mM sodium chloride, 0.05% Tween-20, 0.2% bovine serum albumin, pH 7.4) and add 1 Twelve hours with anti-lumburokinase serum diluted / 5000. The membrane was again washed with blocking buffer and reacted for 90 minutes in blocking buffer in which anti-mouse IgG alkaline phosphotase (AP) -conjugate was diluted to 1/3000. . Finally, by diluting the membrane in the color development solution for 5-10 minutes, the positive plaque portion, which is dark purple on the membrane, was dug up against the agar plate and agar plate of 1 ml SM buffer solution and 50 μl. It was placed in a tube containing chloroform and stored at 4 ℃.

Ii) 2nd, 3rd and 4th screening

In the 2nd, 3rd, and 4th screening, SM plaques containing the positive plaques obtained in the previous screening were diluted to 1 / 100-1 / 1000, respectively, to screen positive plaques in the same manner as in the first step. A total of eight positive plaques were selected until all plaques derived from the plaques were positive.

(2) Rumburokinase coding gene sequence determination

① Lambda DNA Preparation

The eight positive plaque cultures and the host bacterial cultures selected above were incubated for 15 minutes at 37 ° C. in a 1: 400 volume ratio, followed by 80 ml of L-mal-mag-medium (1% tryptone, 0.5% yeast extract, 0.5%). Sodium chloride, 8 mM hydride magnesium sulfide, 10 mM Tris, 0.2% maltose, pH 7.5) and incubated at 37 ° C. for 3 hours. After the cell was cleared and cell debris formed, add 50 μl of chloroform and incubate for another hour to completely disintegrate the bacteria, and then add DNase I and RNase A to the final concentrations of 175 U / mL and 4,550 U / mL, respectively. Incubate for another hour. Sodium chloride was added to the culture solution to the final 1 M, completely dissolved and left for 1 hour on ice, and then the supernatant formed by centrifugation at 12,000 g for 15 minutes at 4 ℃ was transferred to a new tube. Add 0.1 g / ml of polyethylene glycol 8000 (PEG) to the supernatant, dissolve completely, leave for 2 hours in ice, and centrifuge to discard the supernatant and dissolve the precipitate completely with 1 ml of SM buffer. I was. 500 μl of the lysate was transferred to a tube to precipitate polyethylene glycol with chloroform, and the supernatant was taken. EDTA, SDS, and Protease K were added to the supernatant so that their final concentration was 25 mM, 0.32% (w / v), and 0.26 mg / ml, respectively, and then reacted at 65 ° C. for 30 minutes to decompose the capsid protein. Finally, the supernatant was subjected to phenol extraction, isopropanol, and ethanol precipitation to dissolve lambda DNA in the final 40 μl of TE buffer solution. In this way, eight lambda DNAs were obtained from positive plaques.

② Transformation and subcloning of lambda DNA

The eight lambda DNAs obtained above were digested with EcoR I and cut out to identify the rumbaurokinase cDNA fragment inserted through agarose gel electrophoresis. After drilling a small hole in a 0.5 ml tube and clogging with glass wool, a piece of gel was added and the 0.5 ml tube was placed in a 1.5 ml tube and centrifuged several times to elute DNA from the gel. In addition, pGEM-7Zf (+) (Promega) or pGEX-5X-1 plasmid (Pharmacia) was digested with EcoR I and treated with alkaline phosphatase (CIAP: calf intestinal alkaline phosphatase) to prevent self-conjugation. The rumbukinase DNA separated from the plasmid by electrophoresis was conjugated using a T4 DNA conjugated enzyme at a molar concentration ratio of 1: 2. Subsequently, a plasmid conjugated with Lumburokinase cDNA to E. coli JM109 was transduced by heat shock. Subsequently, transformed Escherichia coli was cultured to select plasmid transformants into which the rumbaurokinase cDNA was inserted.

③ Restriction Enzyme Map and Sequence Analysis

The ② the eight kinds of plasmid Nco I (5'-ccatgg-3 ') obtained in the, Kpn I (5'-ggtacc- 3'), BamH I (5'-ggatcc-3 '), Pst I (5 '-ctgcag-3') was digested with 4 restriction enzymes, and the restriction enzyme maps of the individual clones were determined by comparing the positions of the bands on agarose gel electrophoresis, and the clones having the same map were grouped into 3 groups. . Restriction enzyme maps of the respective groups were compared with Lumburokinase-III-1 (U25648) and Lumburokinase-1T4 (U25643) previously known in the GenBank database (http://www.ncbi.nlm.nih.gov). In comparison, one clone that was most similar to rumbaurokinase 1T4 was selected and sequencing was performed to Bioneer Co., Ltd.

In addition, the nucleotide sequences analyzed above were deduced into amino acid sequences through the DNATRANS program (Genome Center / KRIBB, http://genome.kribb.re.kr), and CLUSTA-W for comparison of the respective nucleotide sequences or amino acid sequences. Program (Genome Center / KRIBB, http://genome.kribb.re.kr) was used.

Figure 2 shows the gene sequence of rumbaurokinase (LK-III-2). Lumburokinase III-2 had higher similarity between Lumburokinase 1T4 and amino acid sequence than Lumburokinase III-1.

Example 3

Preparation of Recombinant Expression Vector and Mass Expression of Recombinant Lumburokinase

① Cloning (1) strain and plasmid vector in the expression vector of the Lumburokinase coding gene The strains and plasmids used in this experiment are shown in Table 2 below. system genotype Phenotype Pichia Pastoris GS115 his4 Mut ⁺ E. coli JM109 _{endA1 recA1 gyrA96 thi hsdR17 (r k} -, m k +) relA1 supE44 DELTA (lac-proAB) [F'traD36 proAB lacI q Z M15 DELTA]. Plasmid vector pPIC9K Amp ^r Kan ^r HIS4 E. coli JM109 was used for the maintenance and amplification of the plasmid vector, and Pichia pastoris GS115 ( his 4) (Invitrogen, San Diego, Calif., USA) was used for the expression of rumbaurokinase. (2) Recombination Expression Vector Construction The recombinant expression vector of the present invention was prepared based on FIG. 3. For gene cloning and expression, the shuttle vector pPIC9K provided in the Pichia Pastoris expression kit from Invitrogen was used. In addition, PCR (polymerase chain reaction) was carried out using the rumbaurokinase III-2 gene cloned in the above example to amplify the rumbaurokinase coding cDNA. The two primers (primer) used for PCR were synthesized by Bioneer Corporation (Korea), and the sequence of primers 1 and 2 is shown below. In particular, the recognition site of the restriction enzyme AvrII (underlined italic body) is introduced in primer 2. Primer 1: 5'-cttctcgcccttgcatcgttg-3 'Primer 2: 5'-gt cctagg tagttgttggtaataatgtc-3' PCR reaction is shown in Table 3 below. It is as indicated. [Table 3] matter amount pGEM-7zf (+) / LK III-2 0.1 μg 10 A PCR Buffer 5 μl 50 x dNTP mixtures (10 mM each) 1 μl Primer 1 (10 uM) 1 μl Primer 2 (10 uM) 1 μl Pfu DNA polymerase (0.5 U / μl) 1 μl 3rd sterilized water 40.5 μl PCR reaction conditions were first left for 1 minute at 94 ℃, 35 cycles in order of 30 seconds at 94 ℃, 30 total at 56 ℃, 1 minute at 72 ℃ and finally reacted for 8 minutes at 72 ℃. ② recombinant expression Preparation of vector pPIC9K / LK III-2 The PCR product amplified by the above PCR was treated with restriction enzyme Avr II, and then the reaction was electrophoresed on 1% agarose gel. Thereafter, PCR products were separated with a razor by staining with ethidium bromide (EtBr), followed by pure separation and purification of DNA from agarose gels using a GinClean II kit from BIO101 (vista, CA, USA). Was dissolved again in tertiary sterile water. The rumbaurokinase III-2 PCR product thus prepared was inserted into a purified pPIC9K vector treated with Sna BI and Avr II restriction enzymes, followed by reaction at 4 ° C. for 12 hours. E. coli strain JM109 prepared by the calcium chloride method was transformed with the conjugated reactant, and then transformed onto LB plate medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar) containing 50 μg / ml of ampicillin. Transformants were selected. Primary clones were validated by restriction enzyme treatment and finally transformed with recombinant vector (pPIC9K / LK III-2) and transformant (pPIC9K / LK III-2) inserted with cDNA encoding earthworm rumbaurokinase III-2. ② E. coli JM109). (2) The pPIC9K / LK III-2 vector was transformed with Pichia pastoris, and the pPIC9K / LK III-2 vector was cut and linearized with Sal I, followed by electrophoresis on agarose gel. Pure water was separated, purified and dissolved in sterile water again. The pPIC9K / LK III-2 vector thus prepared was transformed into Pichia pastoris GS115 ( his4 ) using a spheroplast preparation reagent and method of Invitrogen, USA, and the method was as follows. . First obtained by centrifugation of GS115 incubated in a mid-log phase in YPD medium (1% yeast extract, 2% peptone), followed by sterile water, SED (19 ml of 1 M sorbitol, 25 mM EDTA). , 1 ml 1 M DTT), washed with 1 M sorbitol, and then resuspended in 20 ml of SCE (1 M sorbitol, 1 mM EDTA, 10 mM sodium citrate buffer, pH 5.8) solution Divided into dogs. One of these tubes was used to determine the Zymolase titration time, which contained 800 μl of 5% SDS in 200 μl of reactant every 5 minutes for 50 minutes of Zymolase treatment. After transferring to, the turbidity was measured at an optimum absorbance of 800 nm. Depending on the xymolase treatment titration time determined by this method, 7.5 μl of Zymolnez was treated to cells of the other tubes to produce about 70% spheroplasts, which were linearized with Sal I treatment to pPIC9K / LK III -2 was transduced. PPIC9K / LK III-2 linearized with Sal I is inserted at the his4 gene position of the Pichia pastoris GS115 ( his4 ) genome, but the his4 gene on the plasmid is intact and the transformant has a His ⁺ Mut ⁺ phenotype. Will be displayed. Therefore, the transformed yeast cells can be selected on HIS ^- plate medium by expressing the his4 gene included in the pPIC9K vector. The primary screened cells were subjected to secondary screening on YPD plate media (2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml) containing different G418 antibiotic concentrations. The pPIC9K vector contains kanamycin resistance genes, which result in transformed yeast cells that are resistant to G418 antibiotics. High tolerance is shown. As described above, the expression level of the recombinant protein was confirmed by obtaining a high concentration of G418 antibiotic resistance strain through secondary screening.

Recombinant Pichia pastoris strains were selected from 25 ml BMGY medium (1% Bacteria yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.24% yeast nitrate, 4 × ^10-5 % biotin, 1% glycerol ), And then incubated at 240 rpm for 2 days at 240 rpm. The yeast cells were collected by centrifugation in 200 ml BMMY medium (1% Bacteria yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.24% yeast nitrate, 4 × 10 ^-5 % biotin, 0.5% methanol). Resuspension was incubated in a baffle flask for 4 days (240 rpm, 30 ° C.) and methanol was added to the final 0.5% every 24 hours to continuously induce expression. In addition, 1 ml of culture solution was obtained for each time of expression (48 hours, 72 hours, 96 hours), and after centrifugation, only the supernatant was collected and stored at -80 ° C.

Figure 4 is an analysis of the expression pattern of the recombinant rumbaurokinase III-2 protein expressed in yeast transduced with the rumbaurokinase III-2 coding gene of the present invention. 20 μl of each sample of the time-phased culture solution prepared above was mixed with SDS-sample buffer and boiled at 100 ° C. for 10 minutes, and analyzed by 10% SDS-PAGE.

Lane 1: Molecular weight standard protein from top to bottom, 98, 64, 50, 36, 30 kDa.

Lane 2: The transformed yeast clone selected from YPD-G418 plate medium (3.0 mg / ml) was induced to the final 0.5% expression every 24 hours with methanol in BMMY medium, and after 48 hours only the culture supernatant was collected 20 μl was analyzed,

Lane 3: 20 μl of the supernatant of the culture medium was analyzed at 72 hours after the expression of the transformed clone.

Lane 4: 20 μl of the supernatant of the culture was analyzed at 96 hours.

As shown in FIG. 4, the expression level of the recombinant rumburokinase III-2 protein was 96 hours after the induction of methanol in transformed Pichia pastoris selected from YPD medium containing 3 mg / ml G418.

Figure 5 is a analysis of the fibrinolytic activity of the recombinant lumburokinase III-2 protein by the time of the expression as the time of day (48, 60, 72, 96 hours) while inducing the expression by adding methanol to the final 0.5% every 24 hours 5 μl of each obtained culture supernatant was dropped onto fibrin plate medium, and then reacted at 37 ° C. for 2 hours. As a result, the enzymatic titer of the recombinant rumbaurokinase III-2 protein obtained at 96 hours of expression induction was the highest.

As described above, the method for producing rumbaurokinase of the present invention can be obtained by expressing a large amount of recombinant rumbaurokinase III-2 having thrombolytic ability. In addition, the recombinant expression technology can expect various economic effects such as the time and cost of separating and purifying proteins from sources such as earthworms, and the simplification of complex production processes. Can be supplied to the market. In addition, it is possible to completely exclude the possibility of heavy metal contamination that may occur when using the powdered earthworm directly, it can provide a safer roomburokinase with the same enzyme activity as the roomburokinase existing in nature.

In addition, if the recombinant rumbunokinase produced in large quantities in the present invention is applied for therapeutic purposes, it is expected to contribute to the national economy as well as human health by creating a new industry through the development of new drugs.

Claims (6)

(delete) (delete) (delete) Pichia pastoris KFCC-11166 strain expressing Lumbricus rubellus- derived Lumburokinase III-2 protein. delete delete