KR20020014923A - Novel Bacillus sp. for the Stable Expression of Foreign Proteins - Google Patents

Abstract

PURPOSE: Provided is a novel Bacillus sp. whose wprA gene encoding WprA, proteinase of the Bacillus subtilis, is destroyed by homologous recombination to stably express foreign proteins without damaging them. The foreign proteins are produced by transforming the strains with an expression vector and cultured. CONSTITUTION: The preparation method of the Bacillus sp. strain comprises the steps of: constructing gene replacement vector containing wprA gene, Bacillus origin and E. coli origin in which resistant genes to ampicillin and tetracycline are inserted; transforming Bacillus subtilis WB600 using the vector; selecting tetracycline resistant strains and heat-treating them to eliminate the unincorporated vector; screening the genomic DNA of the selected strains by polymerase chain reaction and confirming that wrpA gene is destructed from the genomic DNA of the selected strains; and identifying the strain Bacillus subtilis LB700 (KCTC 18039P) and using it as a host strain to express staphylokinase in a large amount.

Description

Bacillus sp. Microorganism stably expressing foreign protein {Novel Bacillus sp. for the Stable Expression of Foreign Proteins}

The present invention relates to a novel Bacillus sp. Strain that stably expresses foreign proteins. More specifically, the present invention relates to a novel Bacillus sp. Strain in which the gene of the proteolytic enzyme of the Bacillus sp. Strain has been destroyed so as to stably express the foreign protein, and a method for producing a foreign protein using the same.

Bacillus sp. Strain ( Bacillus sp. ) Has a number of advantages, such as a well-developed protein secretion mechanism, the entire gene sequence is known, without causing disease in humans. In addition, because of his endogenous secretion signal sequence, it is known as a very important foreign protein production strain, and unlike other microorganisms have been studied very actively. However, when using strains of the genus Bacillus, not only is the yield of foreign proteins low, but also the phenomenon that the stability of the produced foreign proteins is frequently observed, the main cause of this phenomenon is secreted from the strains of the genus Bacillus itself Degradation of foreign proteins by proteolytic enzymes. For example, staphylokinase, also known as thrombolytics, is produced in very small quantities in Staphylococcus aureus in nature, and there have been attempts to produce it in large quantities in strains of the genus Bacillus (see Behnke, D. and D. Gerlach, Mol. Gen. Genet., 210: 528-534 (1987); Schlott, B., et al., Bio / Technology., 12: 185-189 (1994); Lee, SJ et al., Biotechnol. Lett., 20: 113-116 (1998)), due to the activity of the proteolytic enzymes of the above strain, it was found that the low yield, as well as the stability of the produced staphylokinase.

In order to overcome this drawback, 7 protein hydrolases of Bacillus strains are destroyed, and the staphylokinase gene is placed after the levansucrase promoter and secretion signal sequence, thereby sucrose as sucrose. Although strains have been developed that can induce the expression of kinases (Ye, R. et al., Biotechnol. Bioeng., 62: 87-96 (1999)), the staphylokinases produced therefrom have low stability. The problem could not be solved.

Therefore, there is a constant need to develop strains of the genus Bacillus that can stably express foreign proteins.

Therefore, the present inventors have made intensive research to establish a method for developing a Bacillus strain capable of stably expressing foreign proteins. As a result, the wprA gene, which is a gene encoding WprA, a protease of the Bacillus strain, has been destroyed. Since the foreign strain is expressed in a large amount by transforming the genus strain with the expression vector of the foreign protein, and the foreign protein expressed in the past is stable to proteolytic enzymes, the strain of Bacillus sp. It was confirmed that the foreign protein can be stably produced, and the present invention was completed.

After all, the main object of the present invention is to provide a Bacillus sp. Strain in which the wprA gene of the Bacillus sp. Strain has been destroyed.

Another object of the present invention is to provide a method for transforming an electric strain with an expression vector of a foreign protein, culturing, and preparing a foreign protein therefrom.

1 is a genetic map of the gene exchange vector pDWPRA.

Figure 2 is a graph showing the growth curve of the transformed strain and the expression of staphylokinase according to the culture time.

Figure 3 is a graph showing the change of staphylokinase activity by the protease contained in the culture of each strain.

In the novel Bacillus sp. Strain of the present invention, in order to stably express foreign proteins in the Bacillus sp. Strain, the wprA gene, which is a gene encoding the protein hydrolase WprA, is destroyed so that the WprA is not expressed. It is a constructed strain.

Hereinafter, the strain of the genus Bacillus of the present invention will be described in more detail.

In order to inactivate the wprA gene expressed in the Bacillus subtilis WB600 strain, a gene exchange vector comprising an ampicillin resistance gene, a tetracycline resistance gene, a tetracycline resistance gene in between, a wprA gene, a Bacillus origin and an E. coli origin A gene replacement vector was prepared and used to transform the Bacillus subtilis WB600 strain. The tetracycline resistant strains are then selected and heat treated to remove unincorporated vectors. The genome DNA of the selected strains was screened by polymerase chain reaction (PCR) to confirm that the wrpA gene was disrupted in the genomic DNA of the selected strain, and the strain in which the wprA gene was destroyed in the genomic DNA was referred to as "bacillus." The subtilis LB700 ( Bacillus subtilis, LB700), which was named on August 16, 2000, is a KRIBB Gene Bank (KCTC, 52 Eoeun-dong, Yuseong-gu, Daejeon, Korea). Deposited as KCTC 18039P.

Uses of the Staphylokinase expression vector, including the subtilisin promoter and signal sequence (SPS), the kanamycin resistance gene, the gene encoding staphylokinase (SAK), the Bacillus origin, and the E. coli origin Bacillus subtilis LB700, Bacillus subtilis WB600, Bacillus subtilis DB431 were transformed, and each of them was incubated. As a result of examination of these cultures, it was confirmed that the highest amount of staphylokinase was accumulated in the culture medium of transformed Bacillus subtilis LB700, and the stability of the staphylokinase obtained from the culture medium of the above strains was the best. . From this, it was found that Bacillus subtilis LB700 is a strain capable of stably producing foreign proteins.

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

Example 1 Construction of Bacillus subtilis LB700

In order to disrupt the wprA gene of the Bacillus strain by homologous recombination, a gene replacement vector was constructed as follows: First, the genome of Bacillus subtilis WB600 was used. DNA was used as the template, and the polymerase chain reaction was performed using primers WPR-F: 5'-CCCGAATTCTTGATAGAGCTGGTTTTTTT TATA-3 '(SEQ ID NO: 1) and primers WPR-R: 5'-GCAGAATTCGCACAGCTTCGGCTTATCGGAATT-3' (SEQ ID NO: 2). PCR) was then performed, and the obtained PCR fragment was cut with EcoRI and introduced into pUC19 to construct pUC19-wprA. The electrical vector was cleaved with Bst1107I and EcoRV to obtain a fragment comprising wprA, which was fused with XhoI conjugates. The tetracycline resistance gene obtained by cleaving pUCTV2 with XhoI was inserted at the XhoI position of the fragment containing wprA to obtain a tet-disrupted wprA fragment destroyed by the tetracycline resistance gene, and the electrical fragment was XhoI. PDWPRA, a gene exchange vector, was constructed by inserting into the EcoRI site of pUCTV2 digested (see FIG. 1).

By homologous recombination using pDWPRA, Bacillus subtilis WB600 was transformed, and the transformed strain was cultivated in a solid medium containing tetracycline under a temperature condition of 30 ° C., which was resistant to tetracycline. Was screened and transferred to 5 ml of LB medium at 42 ° C. to remove the unincorporated vector, from which a chromosome was obtained. The electrochromosomes were digested with PvuII, then templated and used with primers WPRP-F: 5'-TGCCAATTGGTTTTCAATTGTTTTAATAGA-3 '(SEQ ID NO: 3) and primer TETP-R: 5'-TAAAATTTGGTTGTGTCGTAAATTCGATTG-3' (SEQ ID NO: 4). PCR was performed. Since the PCR fragment obtained from the wprA gene was included, the gene region encoding the active region of WprA was identified to determine whether the wprA gene was destroyed. That is, by using the hybridization method using the WPRS-F: 5'-ACTTAAATTAATCACCTTTGCTCCTTTGTC-3 '(SEQ ID NO: 5) and WPRS-R: 5'-CTGCGCTGACAGCCTTCATGACATTCA-3' (SEQ ID NO: 6) as a confirmation probe, Searching for the gene region encoding the active site of WprA on the chromosome, it was confirmed that the gene encoding the active site of WprA was destroyed.

Accordingly, the present inventors named the previously selected strain " Bacillus subtilis LB700", and the August 16, 2000, the Korea Institute of Science and Technology (KRIBB) Gene Bank of Korea Institute of Science and Technology (KCTC, 52, Eeun-dong, Yuseong-gu, Daejeon, Korea) was deposited with the accession number KCTC 18039P.

Example 2 Preparation of Staphylokinase

Staphylokinase known to be unstable in Bacillus sp. Strains was prepared from the Bacillus subtilis LB700 (KCTC 18039P) strain as follows: First, the subtilisin promoter and signal sequence of plasmid pKWZ. , SPS) was inserted into the HindIII-EcoRI site of pUC18 as a template, primer SPS-F: 5'-AGCGGATAACAATTTCACACAGGA-3 '(SEQ ID NO: 7) and primer SPS-R: 5'-TTGAGCTCGCCGGCCTGCGCAGACATGTTGCT-3' PCR was performed using No. 8) to obtain SPS fragments to which SacI and NaeI restriction enzyme cleavage sites were added. The electrical sections were cut into EcoRI and SacI, inserted into the EcoRI-SacI site of pUC19, then further cut into EcoRI and PstI site, and then inserted into the EcoRI-PstI site of pKK223-3. Subsequently, pSM704 was constructed by inserting the origin of replication of pUB110 and the kanamycin resistance gene into pKK223-3 containing SPS.

On the other hand, chromosomal DNA of S. aureus (NCTC 10033) was used as a template, and primers SAK-F: 5'-TCAAGTTCATTCGACAAAGGAAAAT-3 '(SEQ ID NO: 9) and primers SAK-R: 5'-GGGAAGCTTATTTCTTTTCTATAACAACCTTTG-3' (SEQ ID NO: PCR was performed using 10) and PCR fragments containing the gene encoding SAK were obtained. The PCR fragment was cut with HindIII and inserted into the NaeI-HindIII site of pSM704 to construct pSAK704, a SAK expression vector.

The constructed pSAK704 was used to transform Bacillus subtilis LB700, DB431 and WB600 of Example 1. Each transformed strain was inoculated into 250 ml of Luria-Bertani culture, followed by incubation at 37 ° C. for 24 hours, and the growth rate of each strain and the expression level of SAK were measured over time (see FIG. 2).

In Figure 2, ( ) Is the growth curve of LB700, ( ) Is the growth curve of DB431, ( ) Is the growth curve of WB600, ( ) Is the amount of SAK secreted in the culture medium of LB700, ( ) Is the amount of SAK secreted in the culture medium of DB431 and ( ) Represents the amount of SAK secreted in the culture medium of WB600. As shown in Figure 2, the growth patterns of the three strains were almost similar, but it can be seen that there is a big difference in the expression pattern of SAK. That is, the expression level of SAK was decreased as time passed by 12 hours peaked in DB431 and WB600, but continued to increase in LB700, after a certain time, the amount was found to be maintained.

Example 3 Stability Test of Staphylokinase

In Example 2, SAK was produced in a large amount in the Bacillus subtilis LB700 strain compared to other strains, which is due to the significantly reduced rate of SAK degradation by WprA, a protease. It was confirmed.

First, E. coli was transformed using another SAK expression vector and cultured, thereby obtaining purely isolated SAK. Meanwhile, Bacillus Bacillus LB700, DB431 and WB600 were incubated at 37 ° C. for 24 hours, and then the culture was centrifuged and filtered to obtain a culture solution. Subsequently, 1 ml of each culture was aliquoted, and 5 mg of purely isolated SAK was added to each of them, followed by standing at 37 ° C. to measure the activity of SAK by standing time (see FIG. 3). The activity of SAK was added to a culture solution containing 40 μl SAK and 40 μl of 10 mM human plasminogen to 900 μl of 20 mM phosphate buffer (pH 7.5), followed by reaction at 25 ° C. for 25 minutes, followed by 20 μl of 50 mM S. -2251 (V-0882, Sigma Aldrich Co., USA) was mixed and determined by measuring absorbance at 450 nm.

Figure 3 is a graph showing the activity of SAK mixed with the culture medium of each strain for each time left when the activity immediately after mixing the SAK and culture medium to 100. As shown in FIG. 3, the activity of SAK mixed with the culture medium of DB431 and WB600 was 50% of the initial activity after 2 hours of incubation, but the activity of SAK mixed with the culture of LB700 was the first activity after 24 hours of incubation. It was found that about 60% was maintained.

As described and demonstrated in detail above, the present invention provides a strain of the genus Bacillus, which is capable of stably expressing foreign proteins, in which the wprA gene of the strain of the genus Bacillus is destroyed. When the foreign protein is expressed using the Bacillus sp. Strain of the present invention, since the expressed foreign protein is stable to the protease of the Bacillus sp. Strain, the foreign protein can be stably expressed.

Having described the specific part of the content of the present invention in detail, for those skilled in the art, such a technology is only a preferred embodiment, the actual scope of the present invention is the appended claims and their equivalents. It will be defined by.

Claims (7)

A Bacillus sp. Strain in which wprA, a gene encoding WprA of the Bacillus sp. Strain, has been destroyed so as to stably express a foreign protein in a Bacillus sp. Strain. The method of claim 1, Destruction of the wprA gene is by homologous recombination Characterized in that Bacillus genus strains. The method of claim 2, Homologous recombination is a gene exchange vector containing a destroyed wprA gene. using a replacement vector) Bacillus genus strains. Bacillus subtilis LB700, KCTC 18039P, in which the wprA gene of Bacillus subtilis WB600 was destroyed. Using the Bacillus sp. Strain of claim 1 as a host microorganism, transforming with an expression vector of a foreign protein, and obtaining a foreign protein from the culture medium cultured therefrom, a foreign protein is produced from the strain of Bacillus sp. How to. The method of claim 5, The host microorganism is Bacillus subtilis LB700, KCTC 18039P) A method for producing a foreign protein in Bacillus strains with the wprA gene destroyed. The method of claim 5, The foreign protein is characterized in that the staphylokinase (staphylokinase) A method for producing a foreign protein in Bacillus strains with the wprA gene destroyed.