KR100532064B1 - Shuttle vector for screening of secretion signal and screening method of secretion signal using the same - Google Patents

Abstract

The present invention relates to a secretion signal search shuttle vector and a secretory signal search method using the same. More specifically, the present invention uses a replication origin, an antibiotic resistance gene, an alpha-amylase gene from which a secretion signal has been removed, a shuttle vector for detecting a secretion signal comprising a promoter of the gene, and a method for searching for a secretion signal using the shuttle vector. It is about.

Shuttle vector for secretion signal search of the present invention can be replicated with each other in two or more strains, there is an effect that can easily screen the fragments having secretion signal activity.

Description

Shuttle vector for screening of secretion signal and screening method of secretion signal using the same}

The present invention relates to a secretion signal search shuttle vector and a secretory signal search method using the same. More specifically, the present invention relates to a secretion signal search shuttle vector including an alpha-amylase gene from which secretion signals have been removed, and a method of searching for secretion signals using the shuttle vector.

With the development of the production technology of useful proteins using microorganisms, mass production of useful medical proteins or industrial enzymes, which had to be obtained in trace amounts in nature, has become possible. Production of useful proteins using microorganisms requires factors such as host microorganisms, genes of interest, vectors, selection markers, promoters and secretion signals. Among these, the secretion signal serves to secrete proteins produced in the cells of microorganisms out of the cells. In the production of recombinant proteins using microorganisms, the secretion of the synthesized useful protein into the periplasmic space or the culture medium has various advantages. Overexpressed proteins can be secreted out of the cytoplasm before they become insoluble aggregates in the cytoplasm, resulting in water-soluble active proteins, and easier to separate and purify proteins secreted out of the cytoplasm than proteins present in the cytoplasm. .

For this reason, researches related to protein secretion in microorganisms have been actively conducted in recent years. The secretion of proteins in bacteria is generally known to be by Sec-dependent transport. This translocation system requires the peripheral membrane protein SecA (SecA), the integral membrane protein SecE, and the integral membrane protein SecE (SecY) genes to secrete proteins. In particular, it is known that the SecY gene is required to secrete proteins in Escherichia coli (W. M. De Vos et al., Genetics and Biotechnology of lactic acid bacteria, BLACKIE ACADEMIC & PROFESSIONAL, 52-103, 1994).

On the other hand, although the amino acid sequence of the secretion signal is known to have a very low conservation region, it is generally a nonpolar amino acid recognized and cleaved by amino acids having 5 to 8 cation side chains, 8 to 15 polar amino acids, and 5 to 8 signal peptidase. It is known to have three distinct domains of G. von Heijine., J. Membr. Biol ., 115, 195-201, 1990.

Prior arts related to secretion signals have been studied a lot of secretion signals from E. coli and yeast. US Pat. No. 5,919,654 discloses a secretion signal gene isolated from yeast, and Korean Patent Registration No. 283677 discloses a newly synthesized secretion signal based on the common characteristics of secretion signals secreted from E. coli. In addition, Korean Patent Publication No. 2003-62854 discloses a secretion type vector which secretes granulocyte-colonal stimulation factor with high efficiency, which includes a complex secretion signal consisting of secretion signals derived from two kinds of yeasts. 5,010,003 and U.S. Patent No. 5,013,652 disclose an invertase secretion signal and an acid phosphatase secretion signal, respectively, as a secretion signal for yeast.

On the other hand, the proteins secreted by the microorganisms each have a specific secretion signal, but the secretion signals known to date are only a fraction. Therefore, in order to produce foreign proteins more efficiently using microorganisms, there is an urgent need to identify various types of secretion signals that secrete proteins with high efficiency.

On the other hand, until now, the production of useful proteins using microorganisms have been mainly focused on the production of pharmaceutical products using E. coli. When producing recombinant protein for food using microorganisms, unlike pharmaceuticals, safety should be considered as the top priority of production efficiency. Therefore, in order to produce food proteins, it is necessary to satisfy the condition that all elements for genetic engineering must be "food products" derived from food microorganisms with proven safety.

Lactobacillus is a GARS (Generally Recognized As Safe) microorganism that has been used in a wide range of foods and has been proven to be the safest strain. Lactobacillus has a plasmid, bacteriophage, transposon, etc., it is possible to develop its own vector for the introduction of the gene, transformation by the electric shock transformation method widely used in the art, and food selection marker genes are also secured Because there is. Also, at all times and for the mass-expression of the foreign protein in lactic acid bacteria useful expression promoter (Jos MBM van der Vossen et al , Appl Environ Microbial, 53, 2452-2457, 1987;...... Teija et al, App Environ Microbial ., 57, 333-340, 1991) and a controllable promoter (Pascalle et al., App. Environ. Microbial ., 62, 3662-3667, 1996).

However, in the production of useful proteins using lactic acid bacteria, studies on the secretion signals are insufficient. Recently, the development of live vaccines using lactic acid bacteria, intestinal carriers of useful hormonal agents, and securing efficient genetic resources and development of lactic acid bacteria insertion vectors have begun in the United States and Europe.

Currently, secretion signals isolated from lactic acid bacteria include PrtP (Kok et al., Appl. Environ. Microbial ., 55, 231-238, 1989; Vos et al. Of serotoprotease derived from Lactococcus lactis Wg2, SK11, NCDO763). , J. Biol Chem, 264, 13579-23585 , 1989;....... Kiwaki et al, Molec Microbiol, 3, 359-369, 1989) and NisP (van der Meer et al, J. Bacteriol, 175 , 2578-2588, 1993), Usp45 of Lactococcus lactis MG1363 (van Asseldonk et al., Mol. Gen. Genet ., 240, 428-434, 1993), PrtM and NisZ (Pugsley, Microbiol. Rev. , 57 , 50-108, 1993), only five proteins are known.

Therefore, the inventors of the present invention, when a fragment having secretory signal activity is inserted into the alpha-amylase gene from which the secretory signal is removed, the alpha-amylase is secreted and the secreted alpha-amylase can be easily observed in phenotype by iodine staining. The present invention was completed by preparing a shuttle vector for detecting secretion signals including alpha-amylase from which secretion signals were removed, and confirming that it is possible to separate various secretion signals from the chromosome of Lactococcus lactis. It was.

Accordingly, an object of the present invention is to provide a shuttle vector for the secretion signal search, including the origin of replication, the antibiotic resistance gene, the alpha-amylase gene from which the secretion signal is removed, and the promoter of the gene.

It is also an object of the present invention to provide a transformant transformed with the shuttle vector.

Another object of the present invention is to provide a secretory signal searching method using the secretory signal searching vector.

In order to achieve the object of the present invention as described above, the present invention provides a shuttle vector for the secretion signal search, including the origin of replication, the antibiotic resistance gene, the alpha-amylase gene from which the secretion signal is removed and the promoter of the gene.

The present invention also provides a transformant transformed with the secretion signal search shuttle vector.

In addition, the present invention comprises the steps of (a) cutting the chromosome of the strain to search for secretion signal with restriction enzyme Pst I and then inserted into the Pst I site of the shuttle vector according to the present invention;

(b) transforming Escherichia coli with the vector of step (a);

(c) culturing the transformed Escherichia coli in step (b) in a solid medium containing soluble starch and antibiotics;

(d) selecting a transformant to form a ring by adding an iodine solution to the solid medium in which the culture of step (c) is completed; And

(e) separating the plasmid from the transformant selected in step (d) and then treating the restriction enzyme Pst I to obtain a fragment with secretory signal activity.

Hereinafter, the present invention will be described in detail.

Shuttle vector for secretion signal search according to the invention is characterized in that it comprises a replication origin, antibiotic resistance gene, alpha-amylase gene from which the secretion signal is removed and the promoter of the gene.

In the present invention, the term "secretion signal" refers to an N-terminal sequence which is cut by a signal peptidase after transporting the protein synthesized in the cell out of the cell. The secretion signal is also called a leader sequence or signal sequence. In this case, transporting the protein out of the cell means secretion into the periplasmic space or the culture medium.

In the present invention, a "shuttle vector" refers to a gene carrier that can be cloned from two or more different bacteria. Preferably, the shuttle vector for detecting the secretion signal of the present invention is a gene carrier capable of mutual replication in Gram-positive bacteria and Gram-negative bacteria, and more preferably, it refers to a gene carrier capable of mutual replication in Escherichia coli and lactic acid bacteria.

As a replication origin included in the secretion signal search shuttle vector of the present invention, it is preferable to use a replication origin of Gram-negative bacteria, a replication origin of Gram-positive bacteria, or a replication origin capable of replicating both Gram-negative bacteria and Gram-positive bacteria. Do. Most preferably, the origin of replication capable of replicating both Gram-negative bacteria and Gram-positive bacteria can be used. Examples of the origin of replication include pWVO1 origin of origin of Lactococcus lactis ssp.cremoris Wg2 (KJ Leenhouts et al., Plasmid , 26, 55-66, 1991), Lactococcus lactis ssp pSH71 origin (NCBI accession no. A09338).

As the antibiotic resistance gene, any known gene commonly used as a selection marker in the art may be used. For example, there are erythromycin resistance genes or chloramphenicol resistance genes. Preferably, an erythromycin resistance gene is used.

In the present invention, the alpha-amylase gene from which the secretory signal has been removed is used as a reporter gene of a search vector for searching the secretory signal. The alpha-amylase gene is a gene encoding an alpha-amylase enzyme that randomly hydrolyzes the internal alpha-1,4-glucoside bond of starch to make a low molecular weight maltodextrin. The alpha-amylase gene that can be used in the present invention is preferably a mature amylase gene fragment that does not contain a secretion signal. Alpha-amylase genes have been cloned in animal cells, including microorganisms, plants, and humans, such as Bacillus sp. And Aspergillus sp. As the alpha-amylase gene that can be used in the present invention, any known type of alpha-amylase gene that does not contain a secretion signal may be used. For example, AMYS (NCBI accession no. P06278), BA-5542 (NCBI accession no. P19531), AMYB (NCBI accession no. Q02906), and the like. Preferably, an alpha-amylase gene (SEQ ID NO: 1) which is cloned from Bacillus lichenifor mis and does not contain a secretion signal may be used.

As the promoter of the alpha-amylase gene in the present invention, any one capable of expressing the alpha-amylase gene can be used. Preferably, a promoter having activity in both Gram-negative bacteria and Gram-positive bacteria is used. Examples of such promoters P23, P23, P2, P _lac , and P _T7 . Preferably, P32 promoters derived from Lactococcus lactis WG2 can be used (Jos MBM van der Vossen et al., Appl. Environ. Microbial ., 53, 2452-2457, 1987).

Preferably, the secretion signal search shuttle vector of the present invention has a cleavage map as shown in FIG. That is, the shuttle vector for detecting the secretion signal of the present invention does not include the secretion signal cloned from pWV01 origin of replication, erythromycin resistance gene (Emr), Bacillus licheniformis , which is the origin of replication of Gram-negative bacteria and Gram-positive bacteria. Alpha-amylase gene (SEQ ID NO: 1) and constant expression P32 promoter for E. coli and lactic acid bacteria.

In one embodiment of the present invention, the pTA322 vector containing the chromosome of Bacillus licheniformis was digested with restriction enzymes Pst I and Hind III to obtain a mature amylase gene fragment (SEQ ID NO: 1) containing no secretion signal. Since there is a restriction enzyme Pst I site between the secretion signal of alpha-amylase gene and mature amylase gene, treatment of the pTA322 vector with restriction enzymes Pst I and Hind III facilitates obtaining a mature amylase gene fragment containing no secretion signal. can do. The mature amylase gene fragment is inserted into the same restriction enzyme site of pMG36e which is a shuttle vector containing erythromycin resistance gene, P32 which is a constant expression promoter for Escherichia coli and lactic acid bacteria, and a replication origin pWV01 which replicates in Gram-negative bacteria and Gram-positive bacteria. Shuttle vector pGS40 for the secretion signal search was prepared (see Fig. 1).

In addition, the present inventors transformed the recombinant vector to Escherichia coli, and then cultured in a medium containing erythromycin to select E. coli transformants resistant to erythromycin, and then, the E. coli MC1061 / pGS40 gene bank of Korea Research Institute of Bioscience and Biotechnology (KCTC) was deposited on September 4, 2003 as accession number KCTC 10517BP.

Accordingly, the present invention provides a transformant transformed with the shuttle vector for detecting the secretion signal of the present invention. Preferably, the present invention provides E. coli MC1061 / pGS40 (Accession No. KCTC 10517BP) transformed with a shuttle vector for secretion signal search of the present invention.

On the other hand, when the fragment having the secretory signal activity is inserted in the front of the alpha-amylase gene of the secretion signal search shuttle vector of the present invention, the amylase synthesized in the cell can be secreted out of the cell. Amylase secreted out of the cells can be easily identified by phenotype using iodine staining. In other words, amylase is an enzyme that hydrolyzes starch into glucose. Thus, when amylase enzyme is not stained by iodine staining after culturing cells transformed with the secretion signal search shuttle vector of the present invention in a solid medium containing starch, It can be confirmed that the secreted starch was hydrolyzed to glucose, and when stained purple, it can be determined that the amylase enzyme is not secreted out of the cell.

Based on the above principle, the present inventors randomly cut a chromosome to search for secretion signal with restriction enzyme Pst I to obtain a secretion signal candidate fragment and then limit the secretion signal candidate fragment to the same restriction of the shuttle vector of the present invention. E. coli was transformed using the enzyme site. The transformants were cultured in a solid medium containing starch and antibiotics and then iodine stained to determine the amylase secretion ability of the secretion signal candidate fragments. The amylase secretion ability was determined that the amylase was not secreted out of the cells when stained with purple as a result of iodine staining, and amylase was secreted out of the cells when forming a ring without staining with purple.

Accordingly, the present invention provides a method for searching for secretion signals using the secretion signal search shuttle vector of the present invention. Secretion signal search method of the present invention is as shown in FIG.

Specifically, the present invention comprises the steps of: (a) cutting the chromosome of the strain to search for secretion signal with restriction enzyme Pst I and then inserted into the Pst I site of the shuttle vector of the present invention;

(b) transforming Escherichia coli with the vector of step (a);

(c) culturing the transformed Escherichia coli in step (b) in a solid medium containing soluble starch and antibiotics;

(d) selecting a transformant to form a ring by adding an iodine solution to the solid medium in which the culture of step (c) is completed; And

(e) separating the plasmid from the transformant selected in step (d) and then treating the restriction enzyme Pst I to obtain a fragment with secretory signal activity.

As the strain to search for the secretion signal in step (a), all kinds of strains may be used, but preferably, strains whose safety has been confirmed to the human body may be used. For example, lactic acid bacteria chromosomes can be used. This is because the secretion signal isolated from the safety confirmed the strain is harmless to the human body because it can be used as an expression vector for food requiring safety.

E. coli transformation in step (b) may use a conventional transformation method known in the art, for example, DEAE-dextran, calcium phosphate method and electroporation method.

The culture of E. coli transformed in step (c) can be carried out in a solid medium for culturing E. coli, including soluble starch and antibiotics. The soluble starch contained in the medium is not particularly limited, but examples thereof include beta-cyclodextrin (β-CD) and pullulan. The content of the soluble starch is preferably 0.5 to 1.0% by weight. The antibiotic may be used as long as it is used as a selection marker of a transformant transformed with a recombinant vector. For example, erythromycin or chloramphenicol can be mentioned. The content of the antibiotic is preferably 100-300 μg / ml. As the solid medium for E. coli culture, those commonly used in the art may be used, for example, LB medium may be used.

In the step (d), by adding an iodine solution to the solid medium, the transformant which forms a ring without staining purple can be easily identified as a phenotype.

In the step (e), the plasmid may be separated from the selected transformant by a conventional method in the art, and the fragment having the secretory signal activity may be isolated by treating the plasmid with restriction enzyme Pst I. As a method for separating the plasmid, an alkali lysis prep, a boiling method, a lithium miniprep method, and the like, which are known in the art, may be used. In addition, as the method for separating the secretion signal fragment from the plasmid can be used a sequencing method, PCR amplification method and restriction enzyme treatment method known in the art.

In one embodiment of the present invention, six types of secretion signals were screened from the chromosome of Lactobacillus Lactococcus lactis LM0230 according to the method of the present invention. That is, the chromosome of Lactococcus lactis LM0230 was randomly cleaved with Pst I to obtain a secretion signal candidate fragment, which was then inserted into the same restriction enzyme site of the shuttle vector according to the present invention, and transformed into E. coli. The E. coli transformants were cultured in a solid medium containing soluble starch and erythromycin, and then six transformants forming a ring were selected by adding iodine solution. In addition, the present inventors separated the plasmid containing the secretion signal from the transformant and named it pGS401, pGS402, pGS404, pGS405, pGS406 and pGS407. The plasmids were each treated with restriction enzyme Pst I to obtain fragments S401, S402, S404, S405, S406 and S407 with secretory signal activity (see Example 2).

Furthermore, the present inventors transformed E. coli with the plasmid containing the secretion signal selected above, and then cultured the transformants to measure amylase secretion ability of each secretion signal. That is, the total fraction and the peripheral cytoplasmic fraction of the transformant were prepared, respectively, and then the total amylase enzyme titer and the peripheral cytoplasmic amylase enzyme titer were measured by known methods, and from the ratio of the peripheral cytoplasmic amylase enzyme titer to the total amylase enzyme titer. The secretion efficiency of the secretion signal of the present invention was calculated. As a result, the secretion signal S405 showed the highest amylase secretion efficiency of 84% (see Example 3).

In addition, the present inventors transformed the lactic acid bacteria with the plasmid containing the secretion signal of the present invention and then examined the secretion ability of the lactic acid bacteria of each secretion signal. As a result of culturing the lactic acid bacteria transformants in a solid medium containing starch and staining with iodine, it was found that all of the transformants formed a ring. Therefore, the secretion signal of the present invention also showed its activity in the lactic acid bacteria, it was found that has the activity of secreting the amylase enzyme out of the cell. In the case of S407 of the secretion signal of the present invention, the alpha-amylase enzyme activity secreted out of the cells by lactic acid bacteria showed a titer of 73.7 U / mg (see Example 4).

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example 1>

Preparation of Shuttle Vector pGS40 for Secretion Signal Search

The alpha-amylase gene, which does not contain a secretion signal, was prepared by partially cutting the chromosome of Bacillus licheniformis with restriction enzyme EcoR I and inserting it into the same site of pBR322 (Kim, IC Ph.D. thesis, isolation of heat-resistant α-amylase and maltogenic amylase genes of Bacillus licheniformis and their enzymatic properties, Seoul National University Press, Seoul, Korea. 1991) were obtained by treatment with Pst I and Hind III.

Alpha-amylase gene (SEQ ID NO: 1) that does not contain the secretion signal obtained above PMG36e (Department of Genetics, Center of) Recombinant vectors were prepared by insertion into the same site of Biological Sciences, University of Groningen, Netherlands (M. Van De Gucht et. Al., Appl. Environ. Microbiol ., 55, 224-228, 1989).

The recombinant vector was transformed into E. coli MC1061 (ATCC 53338). The transformants were cultured in a medium containing erythromycin to select E. coli transformants resistant to erythromycin. The transformed Escherichia coli MC1061 / pGS40 was deposited with the Korea Biotechnology Research Institute Gene Bank (KCTC) on September 4, 2003 as accession number KCTC 10517BP.

The origin of replication pWV01, erythromycin resistance gene (Em ^r ), which is cloned from Gram-negative bacteria and Gram-positive groups by separating the plasmid from the selected E. coli transformants by alkaline lysis, and the amylase gene (α-amy ) And a shuttle vector pGS40 for searching for a secretion signal of 5.3 kb, which includes a constant expression promoter P32 for E. coli and lactic acid bacteria (FIG. 1).

<Example 2>

Search for Secretion Signals in Lactic Acid Bacterial Chromosomes Using Shuttle Vector pGS40 of the Present Invention

Using the shuttle vector pGS40 of the present invention, fragments with secretory signal activity were selected from the lactic acid bacteria chromosome.

First, chromosomal DNA of Lactococcus lactis LM0230 (JD Efstathion et al., J. Bacteriol , 130, 257-265, 1977), a safe lactic acid bacterium, was isolated by modifying a known method (E Johansen et.al., J. Dairy.Sci., 75, 1186-1191, 1992). The cells were recovered by centrifuging 1.5 ml of lactic acid bacteria in M17 medium containing 0.5% glucose at 7,500 rpm for 5 minutes. The recovered cells were frozen for more than 1 hour at -20 ° C and dissolved, and washed with TES buffer consisting of 50 mM Tris (pH 8.0), 1 mM EDTA, and 6.7% sucrose, followed by 25% sucrose and 5% tryptone ( Triton) X-100, 50 mM EDTA, 50 mM Tris (pH 8.0) and 30 mg / ml concentration of lysozyme suspended in 374 μl of STET buffer solution and incubated for 15 minutes at 37 ℃. 40 μl of 20% SDS was added to the culture suspension, mixed, and reacted at 37 ° C. for 30 minutes and at 65 ° C. for 30 minutes. 100 μl of TE (pH 8.0) buffer was added to the reaction solution, the protein was removed by phenol treatment, and 2 times ethanol was added to precipitate the DNA by centrifugation to separate chromosomal DNA. The isolated chromosomal DNA was treated with restriction enzyme Pst I to obtain about 1000 fragments randomly generated by the restriction enzyme.

Each fragment cut by the restriction enzyme Pst I was inserted into the same restriction enzyme site present in front of the amylase gene of the secretion signal search vector pGS40 prepared in Example 1, and then transformed into E. coli MC1061. The transformants were incubated at 37 ° C. for 10 hours in LB solid medium containing 1% soluble starch and erythromycin at 200 μg / ml concentration. After the incubation was completed, iodine solution (0.3% I ₂ + 0.6% KI) was added to the solid medium to express and secrete alpha-amylase to select transformants that form a ring without staining purple. The method of searching for fragments having secretory signal activity according to the present invention as described above is schematically shown in FIG.

As a result, six transformants forming a ring were selected (FIG. 3). Plasmids were isolated from the selected transformants and then named pGS401, pGS402, pGS404, pGS405, pGS406 and pGS407, respectively. Each of the above plasmids was treated with restriction enzyme Pst I to obtain 6 fragments with secretory signal activity, which were named S401, S402, S404, S405, S406, and S407, respectively. Electrophoresis was performed to approximately confirm the characteristics of these secreted signal active fragments. As a result, the fragments selected according to the method of the present invention was found to have a variety of sizes from 1kb to 5kb (Fig. 4).

<Example 3>

Secretion of Escherichia coli of Secretion Signals Selected from Lactic Acid Bacterial Chromosomes

In Example 2, the secretion ability of E. coli of six secretion signals selected from Lactococcus lactis LM0230 was investigated. To this end, E. coli MC1061 (ATCC 53338) was transformed with plasmids pGS401, pGS402, pGS404, pGS405, pGS406 and pGS407 containing the secretion signal of Example 2, and then the total fraction of the transformant and the surrounding cytoplasmic fraction were prepared. Each alpha-amylase activity was measured.

To prepare a total fraction of the transformant, the transformant was inoculated in 5 ml of E. coli medium, LB liquid medium, and incubated at 37 ° C. for 10 hours. After the incubation was completed, 1 ml of the culture was recovered and cell walls were crushed using an ultrasonic crusher on ice. The lysate was then centrifuged at 4 ° C., 6,000 rpm for 5 minutes to obtain a supernatant. The supernatant was used as a sample for measuring the amylase total activity of the whole cell.

In addition, in order to prepare a peripheral cytoplasmic fraction of the transformant, 1 ml of the culture medium of the transformant was recovered and centrifuged at 7,500 rpm for 5 minutes to obtain pellets. The obtained pellet was dissolved in 0.1 M chloride phosphate buffer at pH 7.5 containing 5 mg / ml lysozyme, 10 mM EDTA and 0.3 M sucrose, and then treated at 37 ° C. for 30 minutes. After centrifugation at 4 ° C. and 6,000 rpm for 5 minutes, the supernatant was obtained and used as a sample for measuring the activity of amylase secreted into the surrounding cytoplasm.

The activity of alpha-amylase was measured by DNS assay, a known method (Miller et al .. Anal. Chem. 31. 46-428, 1959). That is, a substrate was prepared by dissolving 1% soluble starch in 0.1 M chloride phosphate buffer, and then 500 µl of the substrate was added to the reaction tube, followed by pretreatment at 55 ° C. The sample prepared above was added to 1 ml, followed by reaction for 30 minutes, and then 3 ml of DNS solution was added to stop the reaction. It was heated for 5 minutes at 90 ~ 95 ℃ and then the absorbance at 575nm to measure the enzyme titer. The secretion efficiency of alpha-amylase was calculated by the ratio of amylase enzyme titer secreted into the surrounding cytoplasm to the total amylase enzyme titer.

As a result of the experiment, the secretion efficiency of the amylase enzyme of the transformant was varied from 12 to 84%. Among them, the secretion signal S405 showed the highest amylase secretion efficiency of 84% (Table 1).

Alpha-amylase Secretion Efficiency in Escherichia Coli of Secretion Signals According to the Present Invention Secretion signal Alpha-amylase titer (U / ml) Secretion efficiency (%) Total potency Peripheral cellular titers S401 2.9 2.0 69 S402 2.8 1.8 64 S404 23.6 4.2 18 S405 2.5 2.1 84 S406 23.7 5.0 21 S407 22.6 2.8 12

In addition, to demonstrate that the secreted enzyme is alpha-amylase, the total fraction of Escherichia coli transformed with pGS405 having the highest secretion efficiency and the surrounding cytoplasmic fraction were developed by SDS-PAGE. It was then stained with Coomassie blue and photographed on an X-ray film. At this time, the total fraction of the transformed E. coli and the surrounding cytoplasmic fraction was used as a control. The bands indicated by SDS-PAGE were identified, and active staining was performed to prove that the bands indicated were alpha-amylases. Active staining was performed according to known methods (Lin LL et al., J. Appl. Microbiol ., 82, 325-334, 1997). After developing by SDS-PAGE, the developed gel was immediately immersed in 50mM Tris-HCl (pH 8.5) buffer containing 1% soluble starch and incubated at 60 ° C. for 2 hours to contain soluble starch. It was made. The cultured gel was immersed in 0.1N KI solution to check whether it was stained purple. In this case, when the alpha-amylase is present in the gel, the soluble starch is decomposed by the alpha-amylase so that it is not dyed purple, and when the alpha-amylase is not present in the gel, the soluble starch is not decomposed and dyed purple.

As a result, it was confirmed that in the total fraction of Escherichia coli transformed with pGS405 and the surrounding cytoplasmic fraction, a band appeared near 55 kDa, the molecular weight of alpha-amylase. In addition, as a result of the active staining of the band, it was confirmed that the alpha-amylase enzyme by confirming the band that is not stained at 55kDa (Fig. 5).

<Example 4>

Secretion of Lactobacillus of Secretion Signals Selected from Lactic Acid Bacteria

4-1) Transformation of Lactic Acid Bacteria Using Plasmids Containing Secretion Signals

In Example 2, the secretion ability of the secretion signal selected from Lactococcus lactis LM0230 was investigated. To this end, the plasmids pGS401, pGS402, pGS404, pGS405, pGS406 and pGS407 containing the secretion signal of Example 2 were replaced with Lactococcus lactis MG1363 (Gasson MJ et al., J. Bacteriol ., 154, 1-9, 1983 (National Institute for Research in Dairying, England, purchased from Gasson MJ) was transformed by electroporation. Preparation of competent lactic acid bacteria during transformation was performed by modifying known methods (Holo. H et al., App. Environ. Microbiol ., 56: 1890-1896, 1989). The Lactococcus lactis MG1363 was inoculated in 5 ml of M17 liquid medium containing 0.5% glucose, incubated at 30 ° C. for 18 hours, and then 50 ml of M17 liquid medium containing 0.5% sucrose, 0.5% glycine, and 0.5% glucose. 1% inoculation was incubated until the absorbance at 600 nm reached 0.5. The cultured cells were collected by centrifugation at 5,000 rpm for 15 minutes at 4 ° C, and the recovered cells were washed twice with washing buffer solution containing 1/10 volume of 0.5M sucrose and 10% glycine. The washed cells were centrifuged in the same manner as above to recover the cells, suspended in 1/100 volume of the wash buffer solution, stored at -70 ° C and used for transformation.

46 μl of competent cells prepared as described above and 100 to 200 ng of plasmid DNA of the present invention were mixed in a 0.2 cm cuvette, followed by electric shock at 2.5 kV and 25 μF using a Gene Pulser system (Bio-Rad). The lactic acid bacteria were transformed by the addition.

4-2) Measurement of secretion ability of secretion signals according to the present invention in transformed lactic acid bacteria

The transformant of Example 4-1) was inoculated into M17 solid medium containing 1% soluble starch, incubated at 30 ° C. for 18 hours, and then iodine solution was added in the same manner as in Example 2 to form a ring. It was confirmed.

In addition, in order to confirm the secretion capacity of each secretion signal after incubating the transformant for 30 hours at 18 ℃ M17 liquid medium containing 0.5% glucose After centrifugation at 7,500 rpm for 5 minutes, the pellet was discarded and only the supernatant was collected. The protein was concentrated by ammonium sulfate precipitation, and the activity of alpha-amylase secreted out of the cells was measured by the DNS measurement method of Example 3 above.

As a result, the transformant including the secretion signal of the present invention was confirmed that the alpha-amylase is secreted to form a transparent ring (Fig. 6). In addition, the alpha-amylase activity of the transformant containing the secretion signal S407 showed a titer of 73.7 U / mg.

As described above, the shuttle vector for secretion signal search according to the present invention can replicate with each other in two or more strains, and can easily screen fragments having secretory signal activity.

Figure 1 shows a schematic cleavage map of the secretion signal search shuttle vector pGS40 of the present invention.

Figure 2 is a schematic diagram showing a method for selecting a secretion signal from the chromosome of Lactococcus lactis using the secretion signal search shuttle vector of the present invention.

Figure 3 transforms Escherichia coli with a secretion signal search shuttle vector comprising a secretion signal candidate fragment obtained from the chromosome of Lactococcus lactis ( Lactococcus lactis ), E. coli transformants containing soluble starch and antibiotics After culturing in a solid medium, iodine solution was added to show a ring-forming transformant.

Figure 4 shows the results of the SDS-PAGE development of secretion signals selected from Lactococcus lactis chromosome in one embodiment of the present invention (lane 1: λ / HindIII size marker, the size from the top 23130, 9416, 6557, 4361, 2322, 2027, 564 bp, Lane 2: S401, lane 3: S402, lane 4: S404, lane 5: S405, lane 6: S406, lane 7: S407).

Figure 5 shows the result of developing the secretion signal S405 having the highest secretion efficiency in E. coli by SDS-PAGE and the result of active staining.

Lane 1: protein marker

Lane 2: total fraction of untransformed Escherichia coli

Lane 3: Peripheral Cytoplasmic Fraction of Untransformed Escherichia Coli

Lane 4: total fraction of E. coli transformed with pGS405

Lane 5: Peripheral Cytoplasmic Fraction of Escherichia Coli Transformed with pGS405

Lane 6: result of active staining of the total fraction of E. coli untransformed

Lane 7: Activation of Peripheral Cytoplasmic Fraction of Untransformed Escherichia Coli

Lane 8: Active staining of the total fraction of Escherichia coli transformed with pGS405

Lane 9: Activation of Peripheral Cytoplasmic Fraction of Escherichia Coli Transformed with pGS405

Figure 6 transforms lactic acid bacteria with a secretion signal search shuttle vector of the present invention comprising a secretion signal candidate fragment obtained from the chromosome of Lactococcus lactis, lactic acid bacteria transformants are cultured in a solid medium containing soluble starch and antibiotics After the addition of iodine solution, the picture shows a transformant that forms a ring.

<110> KIM, Jeong Hwan LEE, Hyong Joo <120> Shuttle vector for screening of secretion signal and screening method of secretion signal using the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1461 <212> DNA <213> bacillus licheniformis <220> <221> gene (222) (1) .. (1461) <223> alpha-amylase <400> 1 gcagcggcgg caaatcttaa tgggacgctg atgcagtatt ttgaatggta catgcccaat 60 gacggccaac attggaagcg tttgcaaaac gactcggcat atttggctga acacggtatt 120 actgccgtct ggattccccc ggcatataag ggaacgagcc aagcggatgt gggctacggt 180 gcttacgacc tttatgattt aggggagttt catcaaaaag ggacggttcg gacaaagtac 240 ggcacaaaag gagagctgca atctgcgatc aaaagtcttc attcccgcga cattaacgtt 300 tacggggatg tggtcatcaa ccacaaaggc ggcgctgatg cgaccgaaga tgtaaccgcg 360 gttgaagtcg atcccgctga ccgcaaccgc gtaatttcag gagaacacct aattaaagcc 420 tggacacatt ttcattttcc ggggcgcggc agcacataca gcgattttaa atggcattgg 480 taccattttg acggaaccga ttgggacgag tcccgaaagc tgaaccgcat ctataagttt 540 caaggaaagg cttgggattg ggaagtttcc aatgaaaacg gcaactatga ttatttgatg 600 tatgccgaca tcgattatga ccatcctgat gtcgcagcag aaattaagag atggggcact 660 tggtatgcca atgaactgca attggacggt ttccgtcttg atgctgtcaa acacattaaa 720 ttttcttttt tgcgggattg ggttaatcat gtcagggaaa aaacggggaa ggaaatgttt 780 acggtagctg aatattggca gaatgacttg ggcgcgctgg aaaactattt gaacaaaaca 840 aattttaatc attcagtgtt tgacgtgccg cttcattatc agttccatgc tgcatcgaca 900 cagggaggcg gctatgatat gaggaaattg ctgaacggta cggtcgtttc caagcatccg 960 ttgaaatcgg ttacatttgt cgataaccat gatacacagc cggggcaatc gcttgagtcg 1020 actgtccaaa catggtttaa gccgcttgct tacgctttta ttctcacaag ggaatctgga 1080 taccctcagg ttttctacgg ggatatgtac gggacgaaag gagactccca gcgcgaaatt 1140 cctgccttga aacacaaaat tgaaccgatc ttaaaagcga gaaaacagta tgcgtacgga 1200 gcacagcatg attatttcga ccaccatgac attgtcggct ggacaaggga aggcgacagc 1260 tcggttgcaa attcaggttt ggcggcatta ataacagacg gacccggtgg ggcaaagcga 1320 atgtatgtcg gccggcaaaa cgccggtgag acatggcatg acattaccgg aaaccgttcg 1380 gagccggttg tcatcaattc ggaaggctgg ggagagtttc acgtaaacgg cgggtcggtt 1440 tcaatttatg ttcaaagata g 1461

Claims (5)

      Shuttle vector for secretion signal search including a replication origin, antibiotic resistance gene, alpha-amylase gene from which secretion signals have been removed, and a promoter of the gene. The shuttle vector pGS40 according to claim 1, having a cleavage map of FIG. 1. The transformant transformed with the shuttle vector for detecting the secretion signal of claim 1. E. coli MC1061 / pGS40 (Accession No. KCTC 10517BP) transformed with a shuttle vector for secretion signal search. (a) cutting the chromosome of the strain to be searched for secretion signal with restriction enzyme Pst I and inserting it into the Pst I site of the shuttle vector of claim 1; (b) transforming Escherichia coli with the vector of step (a); (c) culturing the transformed Escherichia coli in step (b) in a solid medium containing soluble starch and antibiotics; (d) selecting a transformant to form a ring by adding an iodine solution to the solid medium in which the culture of step (c) is completed; And (e) separating the plasmid from the transformant selected in step (d) and then treating the restriction enzyme Pst I to obtain a fragment with secretory signal activity.