KR100212979B1 - Recombinant microorganism which expresses staphylokinase and method of preparing staphylokinase - Google Patents

Abstract

The present invention relates to a staphylokinase expression vector comprising a tac promoter, an ompA signal sequence, and a staphylokinase gene, and E. coli transformed with the expression vector. The transformed E. coli of the present invention accumulates staphylokinase in periplasm and culture.

Description

Staphylokinase-expressing Recombinant Microorganism and Method for Preparing Staphylokinase Using the Same

The present invention relates to a microorganism expressing staphylokinase and a process for preparing staphylokinase using the same. More specifically, the present invention relates to a staphylokinase expression vector and a recombinant microorganism transformed therewith and a method for producing staphylokinase from the recombinant microorganism.

Staphylokinase is a protein that has the function of breaking down blood clots by activating plasminogen in the blood, and thus may be used as a therapeutic agent for stroke and acute myocardial infarction caused by blood clotting in blood vessels. .

It has not been studied in Korea yet, but research is being actively conducted abroad. This study led to the study of mass production of staphylokinase, which has the potential of medical value-added, and the search for a method to be applied academically, clinically, and industrially.

On the other hand, until now, the expression of staphylokinase was uneconomical because it had to undergo a complex purification process to make it into the cytoplasm of E. coli.

Therefore, an object of the present invention is to make the purification easier by allowing the staphylokinase to accumulate in periplasm, which is devised to solve the above problems.

1 is a diagram showing the structure of the staphylokinase expression vector pSAK.

2 is a schematic representation of the process for preparing pSAK vectors.

FIG. 3 is a schematic picture of the skim milk-plasminogen overlay test showing the expression of staphylokinase in recombinant E. coli JM109 strain transformed with a pSAK vector. Control represents recombinant E. coli JM109 strain transformed with pKK-omp vector, and SAK represents recombinant E. coli JM109 strain transformed with pSAK vector.

4 is a gel electrophoresis picture of the periplasmic fraction of recombinant E. coli JM109 transformed with pSAK vector. M is the molecular weight marker and the numbers 0-6 represent the elapsed time after 1PTG induction. SAK stands for Staphylokinase.

In order to achieve the above object, according to the present invention there is provided a staphylokinase expression vector pSAK and E. coli transformed with the expression vector. Staphylokinase expression vector pSAK of the present invention is a tac promoter that is strongly expressed in Escherichia coli, an ompA signal sequence downstream thereof, sak, pSAK encoding staphylokinase, and E. coli Gene ori necessary for replication in E. coli and the gene Amp ^r , a selectable marker of the function of maintaining E. coli in the plasmid (see FIG. 1).

The ompA signal sequence acts to release staphylokinase produced in E. coli cytoplasm through the cell membrane into periplasm. In the present invention, as a gene encoding Staphylococinase, one of Staphylococcus aureus NCTC10033 is used, but one derived from another strain may be used in the same manner.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1

[Preparation of Staphylokinase Expression Vector pSAK]

Staphylokinase expression vector pSAK of the present invention is a tac promoter that is strongly expressed in Escherichia coli, an ompA signal sequence downstream thereof, sak, pSAK encoding staphylokinase, E. coli It consists of the gene ori necessary for replication in the gene and the gene Amp ^r , which is a selection marker of a function of maintaining E. coli in the culture medium (see FIG. 1).

pSAK was prepared by the following process (see Figure 2). First, the plN-III-ompA-Hind vector (Rentier-Delrue, F., Swennen, D. and Martial, J. (1988) Nucleic Acids Res. 16, 8726) was digested with restriction enzymes Xbal and BamHI, followed by Klenow The fragment (Klenow fragment, hereinafter referred to as 'Klenow F') was processed to prepare a DNA of about 80bp including the ompA signal sequence. The pKK223-3 vector (Pharmacia Dong-il Co., Ltd.), which has a strong tac promoter, was treated with HindIII EcoRl and Klenow F. to obtain 4552 bp of DNA. PKK-omp vectors were prepared by binding with ligase. Subsequently, the oligonucleotide (5'GGGAAGCTTCAAGTTCATTCGACAAAGGAAAAT-3 ', 33mer 5'-GGGAAGCTTATTTCTTTTCTATAACAACCTTTG-3', 33mer) was designed from the chromosomal DNA of Staphylococcus aureus NCT1033. The staphylokinase gene obtained by Polymerase Chain Reaction (hereinafter, referred to as 'PCR') was treated with HindIII, and then the pKK-omp vector was combined with DNA obtained by treatment with HinIII to finally prepare pSAK.

Example 2

[Preparation of Staphylokinase Mass Expression Recombinant Microorganism SJL Strain]

Using the staphylokinase expression vector pSAK prepared in Example 1, the recombinant SJL strain of the present invention was prepared by the following method.

First, E. coli JM109 was transformed into easily transformed cells as follows: 50 μl of E. coli JM109 strain shaken in 50 ml of LB medium was inoculated and incubated at 37 ° C. for 2 hours. . Then, cells were obtained by centrifugation, suspended in 16.7 ml of the RF1 solution of Table 1 and left in ice for 30 minutes. This was again centrifuged and suspended by adding 4 ml of the RF2 solution of Table 2 to the precipitated cells, and 50 μl of this was used for transformation.

Transformation was performed as follows:

1 μl of pSAK vector was added to 50 μl of cells that were easily transformed, and left in ice for 10 minutes, and heat shock was applied at 42 ° C. for 90 seconds. 0.5 ml of LB culture solution was added thereto, and incubated at 37 ° C. for 1 hour, and then inoculated into LB-agar medium containing 50 μg / ml of ampicillin and incubated at 37 ° C. for 10 hours. The resulting colonies were confirmed to have been transformed by pSAK and named SJL. The strain was formally named E.coli JM109 / pSAK, and was deposited with KCTC 8775P in the Korea Bank of Science and Technology (KCTC) on January 28, 1997.

Example 3

[Improved Expression and Activity of Staphylokinase from SJL Strains]

200 ml of LB medium was added to a 1-liter beaker, and then 20 ml of the SJL strain of pre-cultured Example 2 was inoculated and cultured in a shaker at 30 ° C. 4 hours after inoculation, isopropyl beta-di-thiogalactopyranoside (lsopropyl-β-D-thiogalactopyranoside (IPTG)) was added at a concentration of 1 mM to induce the expression of staphylokinase. Then, periplasmic fractions were obtained by the following cold osmotic shock technique, and the expression of staphylokinase was confirmed: 1 ml of the culture medium was taken by time after IPTG addition, and centrifuged at 4000 rpm for 1 minute to precipitate cells. And the supernatant was discarded. 20 μl of a STE solution (20% sucrose, 30 mM Tris-Cl pH 8.0, 1 mM EDTA) was added to the precipitated cells, which were allowed to stand on ice for 10 minutes, followed by centrifugation again to obtain a supernatant. The supernatant was expressed by about 50% of all the proteins in the periplasm after gel electrophoresis (Fig. 4) by the laser densitiometer. And when compared with the molecular weight marker it was confirmed that mass production in a yield of about 5μg or more per 1ml culture.

The expressed staphylokinase activity was identified by the following Schkim milk-plasminogen overlay test: The method of this test was as follows. SJL strains were inoculated in LB-agar medium containing ampicillin at a concentration of 50 μg / ml and incubated at 37 ° C. for 10 hours. The upper agar containing Scheme milk (3%), plasminogen (50 μg / ml) and IPTG (1 mM) was then poured into LB-agar medium in which the SJL strain was cultured. After incubation at 37 ° C. for 3 hours after the agar had solidified, a clear area appeared around the strain. However, the recombinant E. coli JM109 strain transformed with the pKK-omp vector without the staphylokinase gene did not produce a transparent region around the strain when subjected to the Schememilk-plasminogen overlay test (see FIG. 3). . This suggests that recombinant staphylokinase activates plasminogen.

As can be seen from the above, in the case of culturing the recombinant microorganism transformed with the novel staphylokinase expression vector pSAK of the present invention, the staphylokinase accumulates in the periplasm, thereby economically mass-producing high purity staphylokinase. .

Claims (4)

Staphylokinase expression vector pSAK comprising a tac promoter, ompA signal sequence, and a staphylokinase gene. Recombinant microorganism E. coli JM109 / pSAK (KCTC 8775P) transformed by the staphylokinase expression vector pSAK of claim 1 to produce staphylokinase. A method for producing high purity staphylokinase comprising the step of culturing the recombinant E. coli of claim 2. Staphylokinase prepared by the step of culturing the recombinant E. coli transformed with the staphylokinase expression vector pSAK of claim 1.

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