KR20010104065A - Lumburokinase protein isolated from a earthworm and overexpression method of lumburokinase protein in yeast - Google Patents

Abstract

The present invention relates to a novel rumburokinase gene sequence and amino acid sequence of an earthworm ( Lumbricus rubellus ), and to a method for mass expression of a rumburokinase gene using a yeast expression system. By using the yeast expression system of the present invention, it is possible to easily obtain a large amount of high-purity recombinant rumbaurokinase protein, and the recombinant rumbaurokinase protein expressed by the present invention increases fibrinosis activity and thus has high medical value.

Description

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

The present invention relates to a method for mass expression of lumpurokinase protein isolated from earthworm and yeast, and more particularly, to the amino acid sequence of lumpurokinase protein having thrombolytic ability isolated from earthworm, rumbu A transformed recombinant host that expresses a nucleotide sequence, an expression vector comprising the same and rumbaurokinase Invention for Pichia pastoris.

Many patents have already been issued for the method of expressing and purifying a desired protein using recombinant technology. In particular, a method for mass expression of proteins using yeast is a host that is more useful because it can be easily purified by secreting the desired protein to the outside. Yeast, Pichia pastoris , grows very fast and can produce a large amount of protein per liter of culture and produces very low levels of its own protein. Instead, it is used in cheap protein-poor medium. It has the great advantage of releasing protein up to grams per liter. In addition, transcription of genes transduced into Pichia pastoris is subject to strict control of the AOX1 (alcohol oxidase gene) promoter, which is rapidly transcribed upon addition of methanol, the substrate of alcohol oxidase. Therefore, studies are currently underway to produce a variety of proteins using Pichia pastoris as a host, and already transformed Pichia pastoris producing a hybrid protein is disclosed in U.S. Patent Nos. 4,683,293, U.S. Patent 4,808,537, U.S. Patent 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 increases with changing living standards and diet is blood flow disease. In particular, myocardial infarction and cerebral hemorrhage are diseases that are rapidly increasing due to the formation of abnormal blood clots in the blood-related diseases, causing various serious social and economic problems in the modern society. The hemostatic system, which is related to blood flow, freely moves blood to blood vessels, and at the same time 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 composition of the hemostatic system can be broadly divided into three groups: blood vessels, platelets, and water-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 used not only for blood coagulation but also for fibrinolysis. Despite the presence of this regulated hemostatic system, blood clot production occurs in the body when thrombogenic stimulation occurs strongly outside. 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 exposing the endothelium to blood vessels. However, depending on the production environment, the composition changes. Venous thrombi consist of red blood cells, large amounts of fibrin, and small amounts of platelets. Arterial thrombi, on the other hand, are primarily composed of platelet aggregation, and fibrin is present in relatively small amounts. These clots cause a variety of diseases, so medically various thrombosis treatments have been developed.

The most commonly used thrombolytic agents so far are 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), histoplasmic phras Minogen activators (tissue-type plasminogen activator; Brott, TG, Haley, EC, and Levy, DE, 1992, Stroke, 23.632) are consumed in large quantities worldwide. However, these thrombolytic agents do not directly dissolve fibrin, the main component of blood clots, but instead activate plasminogen already present in the blood with plasmin and then cause this plasmin Dissolve blood clots by dissolving them. As a result, too much plasmin is formed and often dissolves to the walls of blood vessels, often leading to systemic hemorrhagic disease, which can threaten the lives of many people.

In order to solve this problem, screening from various sources has been performed on a substance directly acting on fibrin to develop a blood clot-specific soluble agent which directly dissolves fibrin and does not attack other proteins in blood vessels relatively well. Currently, much research has been carried out on fibrinase enzymes isolated from various types of 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 atrosa (atrox: Tu, AT, Baker, B., Wongvibulsin, S., Willis, T. 1996. Toxicon, 34 (11), 1295-1300), levetase (Siigur, E , Aaspollu, A., Tu, AT, and Siigur, J. 1996. Biochem Biophys Res Commun, 224 (1), 229-236). In addition to snake venom, Korea also extracted 28.2 kDa protease from Bacillus strain CK 11-4 (Cim. W., Choi, K., Kim, Y., Park, H.). , Choi, J., Lee, Y. , Oh, H., Kwon, I., and Lee, S. 1996. Appl Environ Microbiol, (Tabanus 62 (7), 2482-2488), or the like that feed the bovine blood fulvus ) isolated two proteins (22 kDa and 27 kDa) of the serine protease family having fibrin-independent fibrin-inducing activity.

Earthworms have been widely used as treatments for vascular diseases in China, Korea, and Japan. However, the exact mechanism and its cause are unknown. Recently, six kinds of thrombolytic proteins were isolated from the worm, Lumbricus rubellus , and the scientific name was cited as Lumburokinase (LK). (Mihara, H., Sumi, H., Yoneta, T., Mizumoto, H., Ikeda, R., Seiki, M., and Maruyama, M. 1991. Jap J Physiol, 41, 461-472) Fibrin can be dissolved even in the absence of plasminogen. Table 1 below shows the simple biochemical and physical properties of rumbaurokinases.

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 Has activity above 80 at 37 ° C., pH 2-10; has activity above 60 at 60 ° C., pH 6-9

Lumburokinases are stable to changes in temperature and pH and do not activate plasminogen into plasmin but instead selectively degrade fibrin. In addition, proteins such as albumin or casein, which are not involved in thrombus formation, are not suitable for thrombolysis because they do not degrade. Lumburokinase is currently used as a surface treatment agent for preventing thrombus formation when artificial kidneys are made of urethane. (2) Powder formulations made by drying only the supernatant by grinding earthworms are used as antithrombolytic agents.

In addition, the method for purifying rumburokinase is known from Mihara et al. Of Japan (Japanese Patent Laid-Open No. 58-148824, Japanese Patent Laid-Open No. 59-63184, Japanese Patent Laid-Open No. 59-184131, and Japanese Patent Laid-Open Patent Publication). 64-75431), a new method for separating and purifying lumpurokinase was also developed in Korea. (Domestic Patent No. 92-8369) Recently, DEAE-Sephadex anion exchange column chromatography And p-amino benzamidine affinity column chromatography using a method of easily separating the lumburokinase from the earthworm has been reported, and the cleavage pattern of fibrinogen by rumburokinase and the sequence of the cut site Analysis also revealed 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 very time consuming and expensive to separate and purify rumburokinase directly from earthworms. Instead, the earthworm acquires at least five different proteases in the body at once.

Therefore, the method of refining and separating lumpurokinase directly from the earthworm is not only able to use a single priced and pure species of lumpurokinase due to the cost of the purification and separation process, but the earthworms are polluted in the ground. Risks involving heavy metals.

In order to rectify the above-mentioned problems, the present invention provides a recombinant vector for mass production of a naturally occurring thrombolytic rumbukinase to provide a high yield of lumpurokinase through accurate induction control in transformed host cells. It aims to manufacture.

Accordingly, an object of the present invention is to provide an amino acid sequence of a new lumpurokinase protein in the earthworm ( Lumbricus rubellus ).

It is also an object of the present invention to provide a nucleotide sequence of a gene encoding the above.

In addition, an object of the present invention is to provide a recombinant vector containing the gene of the rumbaurokinase and capable of producing a protein.

In addition, an object of the present invention is to provide a large amount of high-purity recombinant rumbaurokinase having a thrombolytic ability using a recombinant microorganism.

1 is a comparative analysis of the amino acid sequence of Lumburokinase (LK-III-2) of the present invention and the previously known amino acid sequence of Lumburokinase (LK-III-1, LK-1T4),

Figure 2 is a comparative analysis of the nucleotide sequence of the previously known Lumburokinase gene (LK- 1T4 ) and Lumburokinase gene (LK-III-2) of the earthworm ( Lumbricus rubellus ),

Figure 3 shows the schematic diagram of the introduction of the roomburokinase gene into pPIC9K, the yeast expression vector of the present invention,

4 is a recombinant rumburokinase gene expressed in Pichia pastoris in the present invention and confirmed on SDS-PAGE,

5 is a Western analysis of the recombinant rumburokinase of the present invention with anti-lumburokinase,

6 is a measure of the activity of lumburokinase of the present invention in a fibrin plate medium,

Figure 7 shows the final purified recombinant rumbaurokinase protein of the present invention on SDS-PAGE.

In order to achieve the above object, the present invention provides a novel rumburokinase having a thrombolytic ability isolated from the earthworm, the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence SEQ ID NO: 2.

The present invention also provides a recombinant vector capable of expressing rumbaurokinase.

The recombinant vector of the present invention is preferably pPIC9K / LK of FIG.

The present invention also provides a pichia pastoris which is a recombinant host cell expressing the recombinant vector.

Pichia pastoris of the present invention is preferably Pichia pastoris KFCC-11166.

In another aspect, the present invention provides a method for producing recombinant rumbaurokinase comprising culturing the transformed host to induce expression of rumbaurokinase with methanol and to obtain the same.

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 high fibrin decomposition activity fraction. The amino acid sequence of the isolated protein was determined and injected into mice to obtain anti-lumburokinase serum.

In addition, cDNA libraries were prepared by separating mRNAs from the total cells of the earthworms, and the cDNA of rumbaurokinase was cloned using anti-lumburokinase serum. Clones obtained by cloning were analyzed by restriction enzyme map, and among them, clones having the same restriction enzyme map as Lumburokinase-1T4 were selected to determine their sequencing.

The cloned rumbaurokinase was referred to as rumbaurokinase III-2 and inserted into the pPIC9K vector to prepare a recombinant vector pPIC9K / LK.

The pPIC9K vector used in the present invention contains Saccharomyces cerevisiae that secretes expressed proteins out of yeast.Saccharomyces cerevisiae)of Alpha-factor leader sequence (α-factor leader sequence) is included, and His, ampicillin, kanamicin genes are included so that it can be used as a label to confirm the transduction into the host.

BMMY medium, which is a cheap protein-poor medium, was used for the expression of rumbaurokinase in the recombinant host cell Pichia pastoris KFCC-11166 of the present invention, and the mixture was added with 0.5 methanol at 30 ° C. for 48 to 120 hours. Expression was induced. Rumburokinase secreted in the culture solution was easily isolated and purified by performing an affinity column. However, the expression level was different depending on the culture conditions or culture tanks, but the aerobic culture tank was expressed for 96 hours after methanol induction in the aerobic culture medium. 0.1 g of recombinant rumbaurokinase per liter of culture was obtained.

Recombinant Lumburokinase has the same thrombolytic ability as natural Lumburokinase and has a yield of 5 times or more 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 of Lumburokinase from Earthworm and Determination of Amino Acid Sequences of Lumburokinase

(1) Isolation and Purification of Lumburokinase Protein 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 12000 g for 30 minutes at 4 ° C. The supernatant was taken and 0-30 (w / v) ammonium sulfate was added slowly at 0 ° C., then centrifuged, and the precipitate was stored suspended in 50 mM Tris buffer (pH 8.0). The supernatant was again slowly added 30-60 (w / v) ammonium sulphate at 0 ° C. and then centrifuged to discard the supernatant and the precipitate combined with the first precipitate. 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 into fibrin plate media (Astrup, T., and Mullertz, S., 1952. Arch Biochem Biophys, 40, 346-351). Plate medium was measured for 2 hours at 37 ℃ fibrinolytic activity (fibrinolytic activity).

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) and then 20 mM Tris at pH 8.0. Wash with buffer. The elution buffer (500 mM arginine, 20 mM sodium acetate, pH 5.0) was flowed at a flow rate of 1 ml / min, and fractions were collected in 1 ml portions, and 5 µl of the fraction was dropped on the fibrin plate medium. After the reaction for 1 hour at 37 ℃ fibrinolytic activity (fibrinolytic activity) was measured. Based on the measurement results, the fractions with high activity were collected, dialyzed with 50 mM Tris buffer solution at pH 8.0, and concentrated with centriprep (Amicon, USA) to obtain lumpurokinase.

(2) amino acid sequence analysis

The derivatized rumburokinase was denatured by electrophoresis on SDS-PAGE and stained with Coomassie brilliant blue to confirm the position of the rumburokinase, and then the gel of the rumburokinase site was cut and electrolyzed. -eluter: Bio-Rad) was used to elute the protein. 0.4 ml volume was added every 0 min, 10 min, 30 min, 2 h, 6 h, 18 h while reacting at 37 DEG C with the addition of active Lumburokinase or endoproteinase Lys-C to the denatured lumburokinase solution. The reaction was stopped by adding pear SDS-PAGE sample buffer. SDS-PAGE was performed by mass-reacting the samples taken at each time by electrophoresis on SDS-PAGE and mass-reacting in the same manner by selecting the most responsive time among the cut peptides. Fractionated peptides were polyvinidene diflurolide from gels in electroblot buffer (10 mM 3- [cyclohexylamino] -1-propanesulphonic acid (CAPS), pH 11, 10 (v / v) methanol). (PVDF: polyvinyidene difluoride) membrane was moved to 300 mA for 90 minutes and stained to determine the position of the band. Amino acid sequence analysis of each stained band was performed by the Korea Basic Science Institute. The amino acid sequence of the new rumbaurokinase was prepared by SEQ ID NO: 1 and the base sequence was SEQ ID NO: 2.

In FIG. 1, the sequences of amino acids of the new lumpurokinase (LK-III-2) and the existing lumpurokinases (LK-III-1 and LK-1T4) are compared. Although Lumburokinase III-2 showed a large difference from Lumburokinase III-1, only 4 amino acids which were displayed in reverse phase with Lumburokinase 1T4 were inconsistent and all were identical.

Example 2

Preparation of cDNA Library of Lumburokinase Protein and Screening of Lumburokinase Gene

(1) Preparation of Earthworm cDNA Library

① RNA extraction of 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 ℃. Place the frozen earthworm in a mortar and pestle, add liquid nitrogen intermittently, grind finely until it becomes powder, add 100 μl of earthworm powder to the tube, add 1 ml of Ultraspec II RNA Reagent ^?? Melted. After 5 minutes of reaction on ice, 0.2 ml of chloroform was added to the mixture, and the mixture was reacted 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 RNA pellets, and dried. The RNA pellet was dissolved in 50 µl of distilled water mixed with diethyl pyrocarbonate (DEPC) and centrifuged again to take only the supernatant. After reacting for 5 minutes at 65 ° C, RNA was immediately added to the secondary structure. Was not formed. 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 Preparation of oligo dT column

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 phenol extracted, precipitated with ethanol and finally concentrated to 45 μl of solution to obtain mRNA.

④ cDNA synthesis

The primary strand (1 ^st -strand) cDNA and the secondary strand (2 ^nd -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, E. coli DNA polymerase I, and [α- ³² P] dCTP were added to the synthesized primary cDNA strand reactant at 16 ° C. for 5 hours, followed by addition of E. coli DNA conjugated enzyme and T4 bacteriophage polynucleotide kinase. In 30 minutes or at pH 8.0 was added EDTA finally synthesized double stranded cDNA. Double stranded cDNA was phenol extracted and then 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. It was phenol extracted and precipitated with ethanol and suspended in final 29 μl of TE buffer. The suspension was heated at 68 ° C. for 5 minutes, cooled to 37 ° C., and then reacted at 37 ° C. for 30 minutes by adding dNTPs and bacteriophage T4 polymerase. The reaction was then stopped with EDTA, phenol extracted and precipitated with ethanol to suspend cDNA with 13 μl of 10 mM Tris buffer.

⑤ Preparation of Lumburokinase cDNA Library

EcoRI linker was conjugated to cDNA of ④ above. The following reaction mixture was mixed with 13 μl of cDNA solution and reacted at 16 ° C. for 12 hours.

10 × Conjugate Enzyme Buffer 2 μl 500 ng / μl EcoRI linker 2 μl 1 U / μl T4 DNA Conjugase 1 μl 10 mM ATP 2 μl

0.5 μl of the reactant was transferred to a new tube, and 19.5 μl of the reactant was heat treated at 68 ° C. for 15 minutes to stop the reaction, and allowed to stand on ice for 5 minutes, followed by mixing the following solutions for 2 hours at 37 ° C. I was.

10 × EcoRI buffer (H) 20 μl 12 U / μl EcoRI 2 μl Sterile water 150 μl

After completion of the reaction, the cDNA in which the EcoRI fragment was formed was purified and suspended in 20 µl, 10 mM Tris buffer.

To clone the cDNA into lambda bacteriophage, freeze / thaw extract of lambda bacteriophage was removed at -80 ° C, dissolved, and 4 µl of cDNA was added to the ice and left on ice and sonic extract. 15 μl was further added and mixed, followed by reaction at room temperature for 2 hours. To this was added 500 μl of 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, followed by centrifugation for 30 seconds, and then the supernatant. Was taken and stored at 4 ° C.

⑥ titration and amplification of cDNA library

Put 200 μl of E. coli (Y1090, A ₆₀₀ = 0.5) into 6 culture tubes and add 20 μl of the solution diluted 1/10 to 1/10 ⁶ of the cDNA library to each tube for 15 minutes at 3 7 ℃. Incubated. 5 ml NZY upper layer agar (50 ° C., 1 / v) NZ amine (casein hydrolysate), 0.5 (w / v) yeast extract, 0.5 (w / v) sodium chloride, 0.2 (w / v) magnesium hydride, 0.7 (w / v) agar) was added to the tube, mixed with IPTG and X-gal so that the final concentrations were 1.5 mM and 1.2 mM, respectively, and then plated on the lower 1.5 (w / v) NZY agar plate medium. After the upper agar medium was hardened, the cells were incubated at 37 ° C. for 8 hours and the colony color was analyzed to titrate plaque forming units. Based on the titration, 2 × 10 ⁴ pfu / 20 μl and 200 μl of E. coli culture (A ₆₀₀ = 0.5) were mixed in a culture tube and incubated at 37 ° C. for 15 minutes, followed by the addition of 5 ml of NZY upper layer agar medium. Plated on agar plate. After incubation for 4 hours at 37 ℃, when plaque appeared 3 ml of SM buffer solution was poured into the plate medium and reacted for 12 hours at 4 ℃ so that the bacteriophage out of the plate medium and mixed with the SM buffer solution. 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) Screening of rumbaurokinase genes from cDNA libraries

An anti-lumburokinase was prepared that can probe the rumbaurokinase protein for screening the rumbaurokinase gene from the cDNA library.

① Serum Production of Anti-Lumburokinase

The lumpurokinase purified in Example 1 was fractionated on SDS-PAGE, the gel of lumpurokinase (34 kDa) was chopped and the same amount of Freund's adjuvant was mixed with the following solution.

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 pore syringe, and 50 μg of rumbaurokinase was injected per experimental white rat (ICR strain) as described below.

Second injection: 15 days after first injection

3rd injection: 15 days after 2nd injection

4 th injection: 10 days after 3 th injection

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

② Screening of Lumburokinase Genes in cDNA Libraries

Iii) primary screening

The cDNA library was infected with E. coli and cultured at 37 ° C. to show 2 × 10 ⁴ plaques per agar plate medium. When plaques with a diameter of about 1 mm were seen, they were soaked in 10 mM IPTG solution and dried with cellulose membranes on the medium and incubated at 37 ° C. for 12 hours. Mark the same location on the membrane and agar medium with ink, then wash the membrane with blocking buffer (10 mM Tris, 150 mM sodium chloride, 0.05 Tween-20, 0.2 bovine syrum albumin, pH 7.4) and wash the anti-lumburokinase serum. Primary reaction was carried out for 12 hours in primary antibody solution diluted to 1/5000 in blocking buffer solution. The membrane was again washed with blocking buffer and 90 minutes in blocking buffer diluted anti-mouse IgG alkaline phosphotase (AP) -conjugate with 1/3000 dilution. The reaction was secondary. Membranes that have undergone secondary antibody reaction are colored for 5-10 minutes in a color solution, and the positive plaque portion, which develops dark purple on the membrane, is dug up against the agar plate in agar, and then 1 ml of SM buffer The solution was placed in a tube containing 50 μl of chloroform and stored at 4 ° C.

Ii) 2nd, 3rd and 4th screening

In the 2nd, 3rd, and 4th screening, SM buffer containing positive plaques obtained in the previous step screening was 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 found by performing until all the plaques derived from the plaques showed positive.

(2) Determination of Base Sequence of Lumburokinase

① λ DNA Preparation

Eight positive plaque cultures and host bacterial cultures were cultured in a 1: 400 volume ratio at 37 ° C. for 15 minutes, followed by 80 ml of L-mal-mag-medium (1 tryptone, 0.5 yeast extract, 0.5 sodium chloride, under 8 mM hydration). Magnesium, 10 mM tris, 0.2 maltose, pH 7.5) and incubated at 37 ° C. for 3 hours. After the cell was cleared, cell debris formed, 50 μl of chloroform was added and incubated for 1 hour to completely decompose the bacteria. U / mL was added and incubated for 1 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. To the supernatant, polyethylene glycol (PEG: Polyethylene glycol # 8000) was added to a final 0.1 g / ml, completely dissolved, and left for 2 hours in ice. The supernatant was discarded by centrifugation and the precipitate was completely dissolved in 1 ml of SM buffer. 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 respectively added to the supernatant to a final concentration of 25 mM, 0.32 (w / v) and 0.26 mg / ml, 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. In this way, eight lambda DNAs were obtained from positive plaques.

② Transformation and subcloning of lambda DNA

The eight lambda DNAs obtained were digested with EcoRI and cut out by identifying the rumbaurokinase DNA fragment inserted through agarose gel electrophoresis. A small hole was made in a 0.5 ml tube, closed with glass wool, and a piece of gel containing Lumburokinase DNA fragment was added. The 0.5 ml tube was placed in 1.5 ml tube and centrifuged several times to elute DNA from the gel. In addition, self-conjugation was performed by cutting pGEM-7Zf (+) plasmid (Promega) or pGEX-5X-1 plasmid (Pharmacia) with EcoRI and treating calf intestinal alkaline phosphatase (CIAP) extracted from bovine intestine. Prevented. The plasmid and the lumpurokinase DNA isolated by electrophoresis were conjugated in a molar ratio of 1: 2. Subsequently, a plasmid conjugated with Lumburokinase DNA to E. coli JM109 was transduced by heat shock. In addition, the transformed E. coli was cultured to isolate the plasmid.

③ Restriction Enzyme Map and Sequence Analysis

The eight plasmids obtained in ② were NcoI (5'-ccatgg-3 '), KpnI (5'-ggtacc-3'), BamHI (5'-ggatcc-3 '), PstI (5'-ctgcag- The restriction enzyme maps of the clones were determined by comparing the positions of the bands on the agarose gel electrophoresis by cutting with four restriction enzymes of 3 ′), and then, the clones having the same map were grouped into three groups. The maps of these groups were retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov) to find out Lumburokinase-III-1 (U25648) and Lumburokinase-1T4 (U25643). Compared with, one clone most similar to rumbaurokinase-1T4 was selected, and DNA sequence analysis was commissioned to Bioneer Co., Ltd.

The obtained nucleotide sequences were converted into amino acid sequences using the DNATRANS program (Genome Center / KRIBB, http://genome.kribb.re.kr), and the CLUSTA-W program (CLUSTA-W program) was used to compare the respective nucleotide sequences or amino acid sequences. Genome Center / KRIBB, http://genome.kribb.re.kr).

In FIG. 2, the nucleotide sequence of rumbaurokinase (LK-III-2) was compared with rumbaurokinase-1T4. Lumburokinase (LK-III-2) had a high similarity between Lumburokinase-1T4 and the amino acid sequence. Reversed display indicates that the amino acid sequence is different.

Example 3

Recombinant Vector Production and Mass Expression of Recombinant Lumburokinase

① Preparation of recombinant vector pPIC9K / LK plasmid

As shown in FIG. 3, the Lumburokinase III-2 gene in the pGEM-7Zf / LK plasmid prepared in Example 2 was isolated by AvrII restriction enzyme and then conjugated to pPIC9K vector cut by SnaBI and AvrII restriction enzyme. pPIC9K / LK was prepared.

② Transformation of pPIC9K / LK vector into Pichia pastoris

The pPIC9K / LK vector was cut and linearized with SalI (Promega), purified purely with Geneclean II kit (BIO101 comp), and dissolved in sterile water. The pPIC9K / LK vector thus prepared is transformed into Pichia pastoris GS115 ( his4 ) through a reagent and method for manufacturing spirooplast ( Invitrogen ). First, obtain GS115 incubated in a mid-log phase in YPD medium, followed by sterile water, SED (19 ml of 1 M sorbitol, 25 mM EDTA (pH 8.0), 1 ml of 1 M DTT), After washing sequentially with 1 M sorbitol, it was resuspended in 20 ml of SCE (1 M sorbitol, 1 mM EDTA, 10 mM sodium citrate buffer, pH 5.8) buffer and divided into two 10 ml portions. One tube was used to determine the titration time of Zymolase. In the method, the absorbance of 800 nm was measured on a spectrophotometer by adding 800 μl of 5 SDS to the reacted samples at intervals of 5 minutes from before treatment to 50 minutes after treatment with Zymolanes. As a result, the cells of the remaining tubes were treated with 7.5 [mu] l of Zymolanes to produce about 70 spiroplasts. It was transduced with pPIC9K / LK linearized with SalI treatment. Although pPIC9K / LK linearized with SalI is inserted into the his4 site of the yeast GS115 (his4) genome, the his4 plasmid gene is not damaged and the transformant has a His ⁺ Mut ⁺ phenotype. Therefore, the transformed yeast cells can be selected on HIS ^- plate medium by expressing the HIS4 gene included in the pPIC9K vector. Here, the primary screened cells were selected by growing the cells according to the G418 sulfate concentration (2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml) in the medium. The pPIC9K vector contains a kanamycin gene derived from bacteria that makes Pichia pastoris resistant to G418 sulfate antibiotics. When the recombinant gene of pPIC9K / LKIII-2 is transformed into Pichia pastoris, the kanamycin gene is also inserted. As the multiple copies are inserted, the strain becomes resistant even at high concentration to G418 sulfate antibiotic. The multiple copy insertion and the expression level of the protein were not directly proportional but mostly proportional and the strains with high expression level of the recombinant protein were selected by obtaining a strain resistant to high G418 sulfate antibiotics as a secondary strain selection.

?? Expression of Recombinant Lumburokinase in Pichia Pastoris

Clones selected according to G418 sulfate concentration were inoculated in 25 ml BMGY medium (1 bacterium yeast extract, 2 peptone, 100 mM potassium phosphate pH 6.0, 1.24 yeast nitrate group, 4 × 10 ^-5 biotin, 1 glycerol), respectively, 240 Incubated at 30 ° C. for 2 days at rpm. The yeast cells were collected by centrifugation and placed in 200 ml BMMY medium (1 bacterium yeast extract, 2 peptone, 100 mM potassium phosphate pH 6.0, 1.24 yeast nitrate group, 4 × 10 ^-5 biotin) containing 0.5 methanol (Heymann, Germany). Inoculated and incubated for 4 days in a baffle flask at 30 ° C. at 240 rpm to obtain 1 ml of culture each time zone (48 hours, 72 hours, 96 hours). The culture was centrifuged to collect only the supernatant and stored frozen at -80 ° C until the next analysis.

Figure 4 is an analysis of the expression of the recombinant rumbaurokinase protein expressed in yeast transfected with the rumbaurokinase gene of the present invention. 10 μl of the culture sample prepared above was mixed with SDS-sample buffer, and then boiled at 100 ° C. for 10 minutes, and analyzed by 10 SDS-PAGE.

Lane 1: Molecular weight standard protein, from above, 97.4, 66.2, 45.0, 31.0, 21.0, 14.4 kDa.

Lane 2: The transformant of the present invention was screened in YPD-G418 plate medium (1.0 mg / ml), inoculated into BMMY medium, induced with 0.5 every 24 hours with methanol and electrophoresed with 10 μl of supernatant after 96 hours. One,

Lane 3: The transformant of the present invention was screened in YPD-G418 plate medium (2.0 mg / ml), inoculated into BMMY medium, induced with 0.5 every 24 hours with methanol, and electrophoresis was performed 10 μl of the supernatant after 96 hours. One,

Lane 4: The transformant of the present invention was screened in YPD-G418 plate medium (3.0 mg / ml), inoculated into BMMY medium, induced with 0.5 every 24 hours with methanol and electrophoresed 10 μl of supernatant after 96 hours. One,

Lane 5: The transformant of the present invention was screened in YPD-G418 plate medium (4.0 mg / ml), inoculated into BMMY medium, induced with 0.5 every 24 hours with methanol and electrophoresed with 10 μl of supernatant after 96 hours. One,

As shown in FIG. 4, the rumbaurokinase protein was most expressed in the recombinant Pichia pastoris selected in a medium containing 3 mg G418 sulfate per ml of YPD 96 hours after methanol induction.

FIG. 5 is a western blot of anti-lumburokinase and recombinant lumpburokinase expressed from worms purified from earthworms and labeled at 32.4 kDa.

Lane 1: molecular weight standard protein, protein from above having a molecular weight of 98, 64, 50, 36, 30, 16 kDa,

Lane 2: Reaction of the purified lumpurokinase separated from the worm (Lumbricus rubellus ) with anti-lumburokinase diluted to 1 / 3,125,000,

Lane 3: The transformant of the present invention was screened in YPD-G418 plate medium (3.0 mg / ml) and incubated in the medium, induced with 0.5 every 24 hours with methanol, and then expressed with supernatant after 96 hours. Reacted anti-lumburokinase diluted to 1 / 125,000,

Lane 4: The transformants of the present invention were screened in YPD-G418 plate medium (3.0 mg / ml) and incubated in the medium, induced with 0.5 every 24 hours with methanol, and then expressed with supernatant after 48 hours. Anti-lumburokinase diluted to 1 / 625,000.

FIG. 6 is an analysis of fibrin degrading activity of recombinant Lumburokinase III-2 protein by expression time zone, while inducing culture into 0.5 methanol at 48 (1), 60 (2), 72 (3), and 96 (4) time zones. 5 µl each of the obtained supernatant was dropped onto a fibrin plate medium to measure the activity of rumbaurokinase. As a result of fibrin resolution, recombinant rumburokinase III-2 protein activity was highest when 96 hours of methanol was added to the culture medium.

Example 4

Purification and Activity of Recombinant Lumburokinase III-2 Protein

(1) Purification of Recombinant Lumburokinase III-2 Protein

After inoculating the transformant of the present invention into BMMY medium, 0.5 hours every 24 hours with methanol, and 96 hours later, the supernatant was obtained and mixed with ρ-aminobenzamidine resin and stirred for about 12 hours or more. The resin was reacted with the supernatant for at least 12 hours after mounting on the stand so that the column was vertical, and slowly packed the column at a rate of 3 ml / min with a pipette. After the completion of the packing was washed sufficiently with 20 mM tris-hydrochloric acid (pH 8.0). After washing, the elution buffer solution (500 mM arginine, 20 mM sodium acetate, pH 5.0) was flowed at a flow rate of 1 ml / min, and fractions of 1 ml were checked on SDS-PAGE of 12.5.

FIG. 7 shows pure purified Lumburokinase III-2 protein on SDS-PAGE. The 50th fraction showed purity of 95 or more.

Lane 1: Molecular weight standard protein, molecular weight of 97.4, 66.2, 45.0, 31.0, 21.0, 14.4 kDa from above,

Lane 2: 5 µl of the purified solution of the 10th affinity column fraction obtained by culturing the transformant of the present invention and separating recombinant rumbaurokinase by an affinity column,

Lane 3: 5 µl of the purified solution of the 30th affinity column fraction obtained by culturing the transformant of the present invention and separating the recombinant rumbaurokinase by an affinity column,

Lane 4: 5 µl of the purified solution of the 50th affinity column fraction obtained by culturing the transformant of the present invention and separating the recombinant rumbaurokinase by an affinity column,

Lane 5: The transformant of the present invention was inoculated in BMMY medium, and then induced with 0.5 every 24 hours with methanol. After 96 hours, the supernatant was obtained and electrophoresed 50 μl.

As described above, the method of producing lumpurokinase of the present invention can be purified by expressing a large amount of recombinant lumpurokinase having thrombolytic ability. In addition, time and cost are reduced by using recombinant expression technology, thereby providing high purity of rumbaurokinase at a low price.

In addition, the possibility of heavy metal contamination that can occur when using earthworm powder is excluded, and the time is shortened compared to the existing method, and the activity of the protein that can be lowered during the extraction process for a long time, such as a protein purified using recombinant expression technology Lumburokinase is provided with an activity as high as 1.5 times compared to the positive activity.

Claims (6)

Novel lumpurokinase with thrombolytic activity isolated from the earthworm; A nucleotide sequence of SEQ ID NO: 1 encoding the above; And the rumbaurokinase protein comprising the amino acid sequence of SEQ ID NO: 2. A recombinant vector comprising the gene of claim 1 and capable of expressing rumbaurokinase. The recombinant vector of claim 2, wherein the expression vector is a pPIC9K / LK plasmid of FIG. 3. A recombinant host cell expressing the recombinant vector of claim 2. The method of claim 4, wherein the recombinant host cell is Pichia pastoris KFCC-11166. A method for producing a recombinant rumbaurokinase comprising culturing the transformed host of claim 5 to induce the expression of rumbaurokinase with methanol and obtaining the same.