KR20090018315A - Recombinant vector containing ptsl promoter and method for producing exogeneous proteins using the same - Google Patents

Abstract

A recombinant vector containing ptsL promoter derived from Escherichia coli is provided to easily obtain the foreign protein after the stationary growth phase without the cell-crushing process by inducing cytolysis through expression of protein harmful to bacteria or antimicrobial peptide within the cell. The recombinant vector containing ptsL promoter derived from Escherichia coli which contains DNA encoding ptsL promoter activator having the nucleotide sequence of SEQ ID NO:1 and a gene encoding the foreign protein, and optionally a gene encoding the expression regulatory protein of the ptsL gene and the promoter for expressing the expression regulatory protein, wherein the gene encoding expression regulatory protein of ptsL gene is mlc encoding the expression-inhibiting protein of ptsL gene, having the nucleotide sequence of SEQ ID NO:2.

Description

Recombinant Vector Containing ptsL Promoter and Method for Producing Exogeneous Proteins Using the Same}

The present invention relates to a recombinant vector containing an E. coli-derived ptsL promoter and a method for producing a foreign protein using the same, and more specifically, a transformant into which a recombinant vector containing a E. coli-derived ptsL promoter and a gene encoding a foreign protein is introduced. It relates to a method for selectively expressing foreign proteins in the cell growth arrester using.

Escherichia coli coli ) is the most physiological, biochemical, and genetic research so far, particularly useful for producing recombinant proteins or useful metabolites. Production of recombinant protein using E. coli has a number of advantages, such as high production efficiency, shorten time and cost, and occupies an important position in the development of biotechnology. In particular, since DNA and double helix structures have been identified by Watson and Creek, microbial DNA manipulation techniques have been used to produce a variety of useful materials. This DNA manipulation technology has enabled the widespread industrial use of microorganisms, and therefore, technologies for the production of many useful substances have also evolved. Biotechnology, which started with simple protein production, is now available in a wide variety of applications. Application.

Recently, research on overexpression of recombinant proteins based on the basic knowledge of molecular biology and expression technology of proteins has been actively conducted based on the remarkable growth of such biotechnology-related technologies. In the case of overexpression of the recombinant protein, induction expression has been mainly used. Expression systems using T7 promoter, lac promoter, tac promoter and trc promoter induced by isopropyl-β-D-thiogalactopyranoside (IPTG) are applicable. Similar methods are used in protein expression systems other than Escherichia coli. In addition, studies on protein purification techniques for establishing culture conditions for overexpression in benign bacteria, fungi, yeast, animal cells, and the like and for separating the produced recombinant proteins have been conducted in parallel. In addition, it is necessary to secure a strong promoter for overexpression of the recombinant protein, which is not suitable for high concentration culture because most of it is carried out through induction culture. In addition, proteins or antimicrobial peptides that are harmful to cells are difficult to overexpress because they harm E. coli, and the recovery process is complicated and the recovery rate is very low. In order to solve this problem, the use of a stationary expression system without induction culture may facilitate high concentration culture and recovery of the expressed recombinant protein, thereby reducing the time and cost required for protein production.

Conventionally, when recombinant protein is produced using Escherichia coli, the recombinant protein expressed in Escherichia coli is not released into the cell. Thus, the recombinant protein produced in the cell is broken by chemical methods or physical methods using electricity or pressure. Recovered. In addition, if the produced protein or metabolite is toxic to E. coli, there was a problem to find a way to improve it.

Therefore, it is urgent to develop a method for expressing foreign proteins usefully used in various fields of biotechnology, in particular, to understand the physiological and genetic characteristics of Escherichia coli and to easily overexpress harmful proteins without induction culture. There is an urgent need for the development of separation methods.

Accordingly, the present inventors have made diligent efforts to develop a new stopper expression system capable of efficiently overexpressing foreign proteins during the cell growth arrest period. As a result, the present inventors have recombined a gene containing a gene encoding the expression regulatory protein of the ptsL promoter and the ptsL gene. When using the vector, it was confirmed that the foreign protein is overexpressed at the growth arrest stage and the present invention was completed.

The main object of the present invention is to provide a recombinant vector containing the E. coli-derived ptsL promoter and a gene encoding a foreign protein.

Another object of the present invention is to provide a recombinant vector containing an E. coli-derived ptsL promoter, a gene encoding an expression control protein of ptsL gene, a gene expression promoter encoding the expression regulatory protein, and a gene encoding a foreign protein.

Still another object of the present invention is to provide a recombinant microorganism transformed with the recombinant vector and a method for producing a foreign protein in a growth arrest phase by culturing the recombinant microorganism.

Still another object of the present invention is to provide a method for improving a foreign protein using the recombinant vector.

In order to achieve the above object, the present invention provides a recombinant vector containing a gene encoding a ptsL promoter and a foreign protein derived from Escherichia coli .

The invention also, Escherichia coli ) derived ptsL promoter, a gene encoding the expression regulatory protein of the ptsL gene, a promoter for expression of the gene encoding the expression regulatory protein and a recombinant vector containing a gene encoding a foreign protein.

The present invention also provides a recombinant microorganism transformed with the recombinant vector.

The present invention also comprises the steps of culturing the recombinant microorganism to express the foreign protein in the growth arrestor; And it provides a method for producing a foreign protein comprising the step of recovering the expressed foreign protein.

The invention also comprises the steps of (a) constructing a variant library of genes encoding foreign proteins; (b) constructing a genetic recombinant library containing an E. coli derived ptsL promoter and a library of gene variants encoding the foreign protein such that the variant of the foreign protein is expressed by a gene encoding a ptsL promoter; (c) transforming the microbial host cell selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, actinomycetes, yeast and fungi with the genetic recombinant library or the vector comprising the genetic recombinant library; (d) culturing the transformed host cell to express the gene variant library at a cell growth arrester; And (e) screening cells expressing foreign proteins having improved traits.

The invention also comprises the steps of (a) constructing a variant library of genes encoding foreign proteins; (b) an E. coli-derived ptsL promoter, a gene encoding an expression regulatory protein of ptsL gene, a promoter for expressing a gene encoding the expression regulatory protein, such that the variant of the foreign protein is expressed by a gene encoding a ptsL promoter and Preparing a genetic recombinant library containing a library of genetic variants encoding foreign proteins; (c) transforming the microbial host cell selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, actinomycetes, yeast and fungi with the genetic recombinant library or the vector comprising the genetic recombinant library; (d) culturing the transformed host cell to express the gene variant library at a cell growth arrester; And (e) screening cells expressing foreign proteins having improved traits.

According to the present invention, the expression of foreign proteins in the cell growth arrester can produce a high concentration of foreign proteins harmful to cells, and expression of harmful proteins or antimicrobial peptides against bacteria in cells induces cell lysis, thus cell disruption process It is easy to produce foreign proteins or peptides after the growth stop without the need for easy production process, and can be used as a stable expression system that naturally induces extracellular flow, and is introduced with a reporter system to screen various mutant libraries. Useful for such purposes.

The present invention relates to a recombinant vector containing an E. coli-derived ptsL promoter and a gene encoding a foreign protein, and a transformed microorganism into which the recombinant vector is introduced.

In another aspect, the present invention provides a recombinant vector comprising an E. coli-derived ptsL promoter, a gene encoding an expression regulatory protein of ptsL gene, a gene expression promoter encoding the expression regulatory protein, and a gene encoding a foreign protein as an expression promoter; It relates to a transformed microorganism into which the recombinant vector is introduced.

In another aspect, the present invention relates to a method of culturing the transformant to produce a foreign protein in the growth arrest phase.

In the present invention, the ptsL promoter may be characterized in that it comprises a DNA encoding a ptsL promoter activator (nucleotide) having a nucleotide sequence of SEQ ID NO: 1, the gene encoding the expression control protein is a ptsL gene It may be characterized in that the gene encoding the expression inhibitory protein ( mlc ), the mlc gene may be characterized by having the nucleotide sequence of SEQ ID NO: 2.

In the present invention, the gene expression promoter encoding the expression control protein may be characterized in that the BAD promoter, the BAD promoter may be characterized by having a nucleotide sequence of SEQ ID NO: 3.

In the present invention, the foreign protein is a peptide, polypeptide, binding protein, binding domain, adhesion protein, structural protein, regulatory protein, toxin protein, enzyme, inhibitor, hormone, hormone analog, antibody, signal transduction protein, single chain antibody It may be characterized in that any one selected from the group consisting of antigens, cytokines, regulatory factors and parts thereof, the peptide may be characterized in that the antimicrobial peptide, the antimicrobial peptide is buforin II ( Buforin II) or indolicidin (indolicidin).

In the present invention, the microorganism may be characterized in that the Gram-negative bacteria or Gram-positive bacteria, preferably Escherichia coli ).

In the present invention, the microorganism used for the transformation may be modified to not produce intracellular or extracellular protease related to degradation of the foreign protein expressed, in favor of intracellular overexpression of the foreign protein. have.

In the embodiment of the present invention, in order to express the protein in the cell growth arrest phase, the promoter of the expression gene is suppressed in the growth phase (exponential phase), a large amount of foreign protein by the activator in the growth arrest phase E. coli-derived ptsL promoter that can stably express was used. In addition, the ptsL promoter is not expressed in the growth development phase by an expression repressor, but is overexpressed without a derivative while entering the growth stop phase, and thus is particularly effective for overexpressing proteins or antimicrobial peptides harmful to cells.

1 is a schematic diagram showing that the expression of the ptsL gene in E. coli is controlled by a mlc inhibitor and is expressed by a cAMP-CAP activator. That is, when foreign protein expression is regulated by the ptsL promoter in Escherichia coli, it can be seen that protein expression begins by entering the cell growth arrester as shown in FIG. 1 (J. Plumbridge, Mol. Microbiol., 27: 369, 1998).

As a result of confirming the growth curve of Escherichia coli, it was confirmed by two-dimensional gel electrophoresis that the E. coli-derived ptsL gene was expressed in the cell growth arrester, and the foreign protein was transferred to the growth arrester using the ptsL promoter for intracellular overexpression of the foreign protein. Expressed within.

First, in order to secure a ptsL promoter derived from E. coli , ptsL promoter of E. coli K12 (NCBI NC000913) was synthesized using PCR. The synthesized DNA fragment of about 1.5 kb in size was inserted into plasmid pET-22b (+) to prepare a recombinant vector pTSL.

In addition, the recombinant vector pTSL prepared by fusion of an enhanced green fluorescent protein (EGFP) and 6His (six histidine amino acids) as a foreign protein to be expressed in a cell was substituted with restriction enzymes Nde I and Bam. The recombinant vector pPtsL-EGFP-6His was constructed by connecting to the site cleaved with HI.

The recombinant vector pPtsL-EGFP-6His was introduced into E. coli TOP10, and the fluorescence sensitivity of the expressed green fluorescence protein was measured and analyzed by SDS-PAGE gel electrophoresis. It was confirmed that about 28 kDa of green fluorescence protein was overexpressed in the growth stop phase of, and the ratio of the desired protein was about 40 to 60% among total intracellular proteins.

On the other hand, in order to precisely control the expression of the ptsL promoter, it was confirmed whether the mlc gene, which is a gene encoding the expression inhibitory protein of the ptsL gene, and the BAD promoter, a promoter that regulates the expression of the mlc gene, can be used. To this end, the recombinant vector pBAD-mlc was prepared by inserting the mlc gene into the Nco I and Eco RI cleavage sites of the plasmid pBAD / gIII B.

In addition, in order to simultaneously express the ptsL promoter and the mlc gene, a gene fragment containing the mlc gene was inserted from the BAD promoter into the restriction enzymes Pvu II and Sph I cleavage sites of the recombinant vector pPtsL-EGFP-6His prepared above. The combination vector pTSLR-EGFP-6His was produced.

As a result of measuring the fluorescence sensitivity of the expressed green fluorescence protein by culturing the transformant introduced with the recombinant vector pTSLR-EGFP-6His into the E. coli TOP10 strain, the fluorescence sensitivity of the green fluorescence protein increased It was confirmed that the increase. As a result, it was found that the growth arrest expression system by the E. coli-derived ptsL promoter and the gene encoding the expression inhibitory protein of the ptsL gene is a very efficient expression system.

In another embodiment of the present invention, bufoin II, Jenssen et al ., Is an antimicrobial peptide as a foreign protein to be expressed in cells. al ., Clin . Microbiol . Rev. 19: 481, 2006) was inserted into the restriction enzymes Nde I and Bam HI cleavage sites of the recombinant vector pPtsL-EGFP-6His prepared above to construct a recombinant vector pPtsL-BuforinII.

Indolicidin, Jenssen et. al ., Clin . Microbiol. Rev. 19: 481, 2006) was inserted into the restriction enzymes Nde I and Bam HI cleavage sites of the recombinant vector pPtsL-EGFP-6His prepared above to construct a recombinant vector pPtsL-Indolicidin.

mlc In order to confirm the effect of the gene, a recombinant vector pPtsLR-Indolicidin was prepared in the same manner as the method for preparing the recombinant vector pTSLR-EGFP-6His.

The growth curves of the transformants into which the recombinant vectors pPtsL-Buforin II and pPtsL-Indolicidin were introduced were respectively confirmed. The culture medium was collected from E. coli W3110 or Bacillus subtilis DB104 (Kawamura F. et al., J.) . . Bacteriol, 160:. 442-444, was included in the culture medium of 1984), it was confirmed the growth inhibition by the antimicrobial peptide expression level in each. As a result, it was found that the growth rate was slower than that of the control group, which was due to the expressed antimicrobial peptide.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells are E. coli DH5α, E. col JM101, E. coli TOP10, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene), E. coli B, E. coli BL21, and the like. . However, E. coli such as FMB101, NM522, NM538 and NM539 Strains and other prokaryotic species and genera may also be used. In addition, the above-described E. coli, such as Agrobacterium sp, Bacillus subtilis (Bacillus subtilis), such as Agrobacterium A4 Bashile (bacilli), Salmonella typhimurium (Salmonella Another variety of enteric bacteria and Pseudomonas (Pseudomonas) in strains such as typhimurium) or Serratia dry sense (Serratia marcescens) may be used as host cells.

Prokaryotic transformations were described by Sambrook et. al ., the calcium chloride method described in section 1.82 of supra . Optionally, electroporation (Neumann et. al ., EMBO J. , 1: 841, 1982) can also be used for transformation of such cells.

It is apparent to those skilled in the art that the genes used in the present invention may exist independently in a plasmid in a host cell or inserted into a chromosome of a host.

In the present invention, when the foreign protein is expressed in a cell, the gene encoding the foreign protein is one or repeated two or more times, or the gene encoding the foreign protein repeated two or more times is the same or different from each other. This is possible and any combination of the above is possible.

On the other hand, it will be apparent to those skilled in the art that the induction of expression of a foreign protein can be expressed by a suitable other promoter which can induce expression in a growth arrest phase in a host cell.

In another aspect, the present invention relates to a method for improving a foreign protein using the recombinant vector.

In the present invention, the gene recombinant library containing the E. coli-derived ptsL promoter, the gene encoding the expression regulatory protein of the ptsL gene, the gene expression promoter encoding the expression regulatory protein and the gene variant library encoding the foreign protein or By culturing the host cell transformed with the vector comprising the genetic recombinant library, the gene variant library can be expressed in a growth arrester to obtain foreign proteins having improved traits.

The method according to the invention can be applied to all proteins. For example, peptides, polypeptides, binding proteins, binding domains, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, enzymes, enzyme inhibitors, hormones, hormone analogs, antibodies, signal transduction proteins, single chain antibodies, antigens, cytokines, various It can be used for expression and improvement in the growth arrest phase of proteins, including regulatory factors and portions thereof.

Proteins expressed in cells by the method for producing foreign proteins in the growth stop according to the present invention can be present in various forms in the cells, or can be confirmed by expression outside the cells by outflow. If foreign protein is present inside the cells of the microorganism, the primary antibody may be bound to the protein expressed after the cell disruption process, the secondary antibody labeled with a fluorescent compound may be reacted and stained, and then the expression may be observed by color development. It can be confirmed by measuring the activity of the protein or by quantifying each protein. When the expressed foreign protein is leaked to the outside of the cell, it can be observed by the same method as above after separating the cells and solids by centrifugation.

In the foreign protein improvement method according to the present invention, the step of constructing the gene library is DNA shuffling method (Stemmer, Nature , 370: 389, 1994), StEP method (Zhao, H. et. al ., Nat . Biotechnol ., 16: 258, 1998), RPR method (Shao, Z. et. al ., Nucleic Acids Res ., 26: 681, 1998), molecular breeding (Ness, JE et. al ., Nat . Biotechnol. 17: 893, 1999), ITCHY Act (Lutz S. et al ., Cur . Opi . Biotechnol ., 11: 319, 2000), error-prone PCR (Cadwell, RC et al ., PCR Methods Appl ., 2:28, 1992), point mutations (Sambrook et al ., Molecular Cloning ; A Laboratory Manual , Cold Spring Harbor , NY, 1989) and the like can be obtained by mutating a gene encoding a wild type foreign protein, but is not limited thereto.

In the present invention, the screening step may be characterized by using a protein that recognizes the activity of the foreign protein, a substance labeled on the foreign protein, a labeled ligand that binds to the foreign protein, or an antibody that specifically binds to the foreign protein. Flow cytometry (Georgiou, G., Adv) Protein Chem ., 55: 293, 2000). In the case of using the activity of the protein can be screened by measuring the growth of the host in which the protein is expressed, or by measuring the color reaction catalyzed by the protein.

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 to be construed as limited by these examples.

Example  1: Sample Sampling and Analysis According to Growth Curve of Escherichia Coli

4 representative E. coli W3110, E. coli BL21 (DE3), E. coli HB101 and E. coli XL1-Blue) were selected and cultured in LB liquid medium (a in FIG. 2) and R / 2 liquid medium (b in FIG. 2), respectively.

Each sample was obtained during the exponential growth phase and the growth stop phase, and the cells were disrupted using an ultrasonic crusher, and then two-dimensional gels were prepared using PROTEAN IEF cells and PROTEAN II xi cells (Bio-Rad Laboratories Inc., Herculules, CA, USA). Electrophoresis was performed.

Two-dimensional gel electrophoresis is a method of developing whole proteins in a cell by using a difference in molecular weight and amount of charge, which are inherent to proteins in two-dimensional space (Hochstrasser, et. al ., Anal. Biochem ., 173: 424-35, 1988; Han, et al., J. Bacteriol ., 183: 301-8, 2001), the cell culture was centrifuged at 6000 rpm for 5 minutes at 4 ° C, and then the supernatant was discarded and 500 µL of low salt buffer solution (KCl 3 mM). , KH ² PO ⁴ 1.5 mM, NaCl 68 mM, NaH ² PO ^{4 9} mM), the remaining medium of the precipitate was washed. The precipitate was suspended in 100 μl cell lysis buffer (cell lysis buffer; urea 8M, CHAPS 4% (w / v), DTT 65mM, Tris 40mM), and then centrifuged at 12,000 rpm for 10 minutes at 4 ° C for total protein. The protein was quantified using Bradford method (Bradford MM, Anal. Biochem ., 72: 248-254, 1976).

200 μg of the protein is 340 μl IEF denaturing solution (urea 8M, CHAPS 0.5% (w / v), DTT 10mM, bio-lyte (pH 3-10) 0.2% (w / v), bromophenol blue ) Dissolved in 0.001% (w / v)) and then hydrated in 17 cm ReadyStripTM IPG Strips (pH 3-10, Bio-Rad Laboratories Inc., Herculules, CA, USA) for 12 hours at 20 ° C Isoelectric focusing was performed. The strip is then shaken for about 15 minutes in equilibration buffer I (urea 6M, SDS 2% (w / v), Tris-HCl (pH 8.8) 0.375M, glycerol 20% (v / v), DTT 130mM). After immersion, again equilibration buffer II (urea 6M, SDS 2% (w / v), Tris-HCl (pH 8.8) 0.375M, glycerol 20% (v / v), iodoacetamide 135 mM, bromine After dipping for 15 minutes in a biphenol blue 3.5M), the strip was placed on an SDS-PAGE gel to perform protein separation according to molecular weight.

The protein was stained with a silver staining kit (Amersham Biosciences, Uppsala, Sweden) and the two-dimensional gel was scanned with a GS710 Calibrated Imaging Densitometer (Bio-Rad Laboratories Inc., Herculules, CA, USA), Melanie II (Bio (Rad Laboratories Inc., Herculules, CA, USA) software to determine the number of proteins or spot numbers on the gel.

3 and 4 show E. coli W3110 ( F-mcrA mcrB IN (rrnD-rrnE) 1- ) and E. coli BL21 (DE3) (F - ompT hsdSB (rB - mB -) gal dcm ( DE3 )) shows the results of two-dimensional gel electrophoresis.

In addition, in order to identify proteins with higher expression levels than the exponential growth phase in the growth stop phase, proteins that were not expressed in the exponential growth phase were selected as candidate groups, and the protein expression rate of each candidate group was measured by MALDI analysis. (FIG. 5). Figure 5 A shows the expression rate of the protein expressed in the growth stop of E. coli W3110, Figure 5 B shows the expression rate of the protein expressed in the growth stop of E. coli BL21 (DE3). As a result, it was confirmed that the ptsL gene is expressed in the growth arrest phase.

Example 2 Construction of Recombinant Vector pTSL

In order to obtain a ptsL promoter derived from E. coli , a chromosomal DNA of E. coli K12 (NCBI NC000913) was used as a template, and a ptsL promoter (SEQ ID NO: 1) was synthesized by PCR using primers of SEQ ID NO: 4 and SEQ ID NO: 5. It was.

SEQ ID NO: 5'-GTA GGC ATG CAC CGA TGA TTT CAT CCA GTT CA-3 '

SEQ ID NO: 5'-CCC ACT CCA TAT GTT GCT ACC TCC TTT ATT ATC GT-3 '

PCR conditions consisted of the first denaturation (7 min at 94 ° C), the second denaturation (1 min at 94 ° C), hybridization (1 min at 55 ° C), and extension (1 min 30 sec at 72 ° C) The last extension was carried out at 72 ° C. for 7 minutes.

The plasmid pET-22b (+) (Novagen) isolated from the obtained 1.5 kb DNA fragment was digested with restriction enzymes Sph I and Nde I, and then digested with the same restriction enzymes Sph I and Nde I. , USA) using T4 DNA ligase. The reaction dielectric was introduced into E. coli TOP10 (Invitrogen, USA) using the electroporation method. The transformed strain was selected in LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pTSL (FIG. 6).

Example 3: Construction of recombinant vector pPtsL-EGFP-6His

In order to confirm the expression of the protein in the growth arrester cells by the ptsL promoter, a recombinant vector was constructed to express a gene in the form of an fusion of enhanced green fluorescent protein (EGFP) and 6His (six histidines). .

In order to fuse 6His to the C-terminus of the green fluorescent protein gene, PCR was performed using primers of SEQ ID NO: 6 and SEQ ID NO: 7.

SEQ ID NO: 5'-GGA ATT CAT GGT GAG CAA GGG CGA GGA G-3 '

SEQ ID NO: 5'-TTG GAT CCT TAG TGG TGG TGG TGG TGG TGC TTG TAC AGC TCG TCC-3 '

PCR conditions were performed 30 times of the first denaturation (5 minutes at 94 ℃), the second denaturation (45 seconds at 94 ℃), hybridization (45 seconds at 56 ℃), extension (45 seconds at 72 ℃), The last extension was carried out at 72 ° C. for 7 minutes.

The multi-cloning site of the pTSL recombinant vector prepared in Example 2 includes restriction enzymes Nde I and Bam HI cleavage sites, which encode foreign proteins to be expressed on the cell surface. The gene was inserted. That is, the DNA fragment of about 800 bp size obtained as the PCR product was isolated and digested with restriction enzymes Nde I and Bam HI, and the recombinant vector pTSL prepared in Example 2 was subjected to the same restriction enzyme Nde I as described above. And Bam HI, and then linked using T4 DNA ligase. The reaction dielectric was introduced into E. coli TOP10 using the electroporation method. The transformed strain was selected in LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pPtsL-EGFP-6His (FIG. 7).

Example  4: ptsL  Expression of Recombinant Proteins by Promoters

Recombinant vector pPtsL-EGFP-6His prepared in Example 3 E. coli TOP10 (F- ara D139 Δ ( ara leu ) 7697 Δ lacX74 galU galK rpsL deoR Φ 80d lacZ Δ M15 endA1 nupG recA1 mdrA Δ ( mrr hsdRMS mcrBC )) to obtain the transformed microorganism.

In order to confirm the expression of the fusion protein of green fluorescent protein (EGFP) and 6His by the ptsL promoter, the transformed E. coli TOP10 was inoculated in LB liquid medium containing 2% (w / v) glucose and incubated at 37 ° C. A certain amount of the culture solution was obtained over time to measure the fluorescence sensitivity of the expressed fluorescent protein (FIG. 8). The recombinant vector pPtsL-EGFP-6His has a ptsL promoter, and the gene was expressed at the growth arrest phase according to the extent of cell growth.

In addition, the recombinant vector pPtsL-EGFP-6His was transformed into E. coli. After culturing the transformants introduced in the TOP10, and extracting a certain amount of the culture solution over time to concentrate the expressed green fluorescent protein by 5 times, 40ul each was taken to the SDS-PAGE sample buffer (Tris-HCl 60mM, 25% glycerol , 2% SDS, 2-mercaptoethanol 14.4 mM, 0.1% bromophenol blue) was mixed with 10 µl of water in boiling water for 10 minutes, followed by 12% SDS-PAGE gel electrophoresis. After the electrophoresis gel was soaked in a dyeing solution (coomassie brilliant blue R 0.25g / L, methanol 40%, acetic acid 10%) for 2 hours or more, the gel was decolorized solution (methanol 10%, acetic acid 10%) 2 It was soaked twice over time for discoloration.

As a result, the ptsL promoter overexpressed about 28 kDa fluorescent protein at the growth stop phase of Escherichia coli, and it was found that the ratio of the desired protein was about 40 to 60% among the total intracellular proteins (Fig. 9).

Example  5: recombinant vector pBAD - mlc Made of

For precise regulation of the expression of the ptsL promoter, the mlc gene, a gene encoding a protein whose expression is regulated by the BAD promoter and inhibits the expression of the ptsL gene, was used.

The genomic DNA of E. coli K12 was used as a template, and the mlc gene (SEQ ID NO: 2) was synthesized by PCR using primers of SEQ ID NO: 8 and SEQ ID NO: 9.

SEQ ID NO: 5'-CAT GCC ATG GTT GCG GAA AAT CAG CCT G-3 '

SEQ ID NO: 5'-GGA ATT CTT ATT AAC CCT GCA ACA GAC GAA-3 '

PCR conditions consisted of 30 steps of the first denaturation (5 minutes at 94 ° C), the second denaturation (50 seconds at 94 ° C), hybridization (50 seconds at 55 ° C), and extension (1 minute and 10 seconds at 72 ° C). The last extension was carried out at 72 ° C. for 7 minutes.

The DNA fragment of about 1,221 bp obtained above was isolated. Since the restriction enzyme Nco I site is present at the 5'-end of the mlc gene and the Eco RI site is present at the 3'-end, the DNA fragment is cleaved with restriction enzymes Nco I and Eco RI, and the cleaved DNA fragment is The pBAD / gIII B plasmid digested with the same restriction enzymes Nco I and Eco RI (Invitrogen, USA) was linked using T4 DNA ligase and introduced into E. coli TOP10 using the electroporation method. . The transformed strain was selected in LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pBAD-mlc (FIG. 10).

Example  6: recombinant vector pTSLR - EGFP -6 His Made of

In order to prepare a recombinant vector capable of confirming the expression of the ptsL promoter and the mlc gene at the same time, the primers of SEQ ID NO: 9 and SEQ ID NO: 10 from the BAD promoter to the terminator of the recombinant vector pBAD-mlc prepared in Example 5 were used. The harvest was carried out by PCR.

SEQ ID NO: 10'5-CGC CAT GCA TGC AAG AAA CCA ATT GTC CAT ATT-3 '

PCR conditions consisted of 30 steps of the first denaturation (5 min at 94 ° C), the second denaturation (50 sec at 94 ° C), hybridization (50 sec at 56 ° C), and extension (1 min 30 sec at 72 ° C). The last extension was carried out at 72 ° C. for 7 minutes.

DNA fragments of about 1,600 bp in size obtained above were isolated, digested with restriction enzyme Sph I, and recombinant vector pPtsL-EGFP-6His prepared in Example 3 was digested with restriction enzymes Pvu II and Sph I. , T4 DNA ligase (ligase) was linked, and introduced into E. coli TOP10 by using an electroporation method (electroporation). The transformed strain was selected from LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pTSLR-EGFP-6His (FIG. 11).

Example  7: Expression of recombinant protein at growth arrest

After introducing the recombinant vector pTSLR-EGFP-6His prepared in Example 6 into E. coli TOP10 to obtain a transformed microorganism, the transformant was cultured to measure the fluorescence sensitivity of the expressed fluorescent protein over time. .

That is, after culturing the strain transformed with the recombinant vector pTSLR-EGFP-6His in the same manner as in Example 4, 1mL of the culture medium was centrifuged for 5 minutes at 6000rpm at 4 ℃ according to the culture time, the supernatant was discarded Washed twice with TE buffer solution and then suspended in TE buffer solution again. The cell mixture was analyzed with a fluorometer (Fluorospectrophotometer, SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA). The sample was excited by a 485 nm helium / neon laser and the image was filtered by a 512 nm filter.

As a result, it was confirmed that the fluorescence was weak at the beginning of the culture, but the fluorescence degree was increased while entering the growth stop phase, indicating the highest sensitivity (FIG. 12). This means that the foreign protein expressed by the ptsL promoter was specifically overexpressed in the cell growth arrest phase of E. coli. Therefore, it was found that the growth stop expression system including the ptsL promoter, BAD promoter and mlc gene of E. coli is a very efficient expression system.

Example 8 Construction of Recombinant Vector pPtsL-Buforin II

In order to confirm the expression level of the antimicrobial peptide in the growth arrester cells of the ptsL promoter, a recombinant vector comprising a buforin II gene (SEQ ID NO: 11) was prepared as an antimicrobial peptide.

For the expression and secretion of buforin II antimicrobial peptides with the correct amino acid sequence, trimethyl can be secreted into the periplasmic space via a twin arginine translocation (tat) -dependent pathway at the N-terminus. The amine N-oxide reductase (TorA) secretion signal gene was fused by PCR using primers of SEQ ID NO: 12 and SEQ ID NO: 13.

SEQ ID NO: 12'-AGA TAT ACA TAT GAA CAA TAA CGA TCT CTT T-3 '

SEQ ID NO: 5'-TTG GAT CCT TAT TAT TTA CGC AGC AGA CGA TGA ACA CGA CCC ACC GGA AAC TGC AGA CCC GCA CGG CTG CTA CGG GTC GCA GTC GCA CGT CGC-3 '

PCR conditions were performed 30 times of the first denaturation (5 minutes at 94 ℃), the second denaturation (50 seconds at 94 ℃), hybridization (50 seconds at 56 ℃), extension (50 seconds at 72 ℃), The last extension was carried out at 72 ° C. for 7 minutes.

The DNA fragment of about 200 bp size obtained above was isolated and cut with restriction enzymes Nde I and Bam HI, and the recombinant vector pPtsL-EGFP-6His prepared in Example 3 was prepared with the same restriction enzymes Nde I and Bam as described above. After cleavage with HI, linkage was made using T4 DNA ligase. The reaction dielectric was introduced into E. coli TOP10 using the electroporation method. The transformed strain was selected from LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pPtsL-Buforin II (FIG. 13).

Example  9: recombinant vector pPtsL - Indolicidin Made of

In order to confirm the expression level of the antimicrobial peptide in the growth arrester cell of the ptsL promoter, a recombinant vector including an indolicidin gene (SEQ ID NO: 14) was prepared as an antimicrobial peptide.

For the expression and secretion of indolisidine antimicrobial peptides with the correct amino acid sequence, trimethyl can be secreted into the periplasmic space via a twin arginine translocation (tat) -dependent pathway at the N-terminus. The amine N-oxide reductase (TorA) secretion signal gene was fused by PCR using primers of SEQ ID NO: 12 and SEQ ID NO: 15.

SEQ ID NO: 15'-TTG GAT CCT TAT TAA CGA CGC CAC GGC CAC CAC GGC CAT TTC CAC GGC AGA ATC GCA GTC GCA CGT CGC GGC-3 '

PCR conditions were performed 30 times of the first denaturation (5 minutes at 94 ℃), the second denaturation (50 seconds at 94 ℃), hybridization (50 seconds at 56 ℃), extension (50 seconds at 72 ℃), The last extension was carried out at 72 ° C. for 7 minutes.

DNA fragments of about 184 bp size obtained above were isolated and digested with restriction enzymes Nde I and Bam HI, and the recombinant vector pPtsL-EGFP-6His prepared in Example 3 was subjected to the same restriction enzymes Nde I and Bam as described above. After cleavage with HI, linkage was made using T4 DNA ligase. The reaction dielectric was introduced into E. coli TOP10 using the electroporation method. The transformed strain was selected from LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pPtsL-Indolicidin (FIG. 14).

Example 10 Construction of Recombinant Vector pPtsLR-Indolicidin

In order to prepare a recombinant vector capable of confirming the expression of the ptsL promoter and the mlc gene at the same time, the primers of SEQ ID NO: 9 and SEQ ID NO: 10 from the BAD promoter to the terminator of the recombinant vector pBAD-mlc prepared in Example 5 were used. Obtained by the PCR method.

PCR conditions consisted of 30 steps of the first denaturation (5 min at 94 ° C), the second denaturation (50 sec at 94 ° C), hybridization (50 sec at 56 ° C), and extension (1 min 30 sec at 72 ° C). The last extension was carried out at 72 ° C. for 7 minutes.

DNA fragments of about 1.6 kb obtained above were isolated, cut with restriction enzyme Sph I, and the recombinant vector pPtsL-Indolicidin prepared in Example 9 was cut with restriction enzymes Pvu II and Sph I, followed by T4. Linking was done using DNA ligase. The reaction dielectric was introduced into E. coli TOP10 using the electroporation method. The transformed strain was selected in LB plate medium to which antibiotic ampicillin (100 μg / L) was added to obtain a recombinant vector pPtsLR-Indolicidin (FIG. 15).

Example 11 Cultivation of the transformants into which the recombinant vectors pPtsL-BuforinII and pPtsL-Indolicidin were introduced

The antimicrobial peptide expression recombinant vectors pPtsL-Buforin II and pPtsL-Indolicidin prepared in Example 8 and Example 9 were introduced into E. coli TOP10 to prepare a transformant. The transformants were shaken using BioScreen C (Oy Growth Curves Ab Ltd., Helsinki, Finland) for 30 hours at 30 ° C. in MaxyBroth liquid medium.

As a result, the growth curve of the buforin II-expressing cells was similar to that of the control group, but the indolicidin-expressing cells showed relatively slow growth rate and were markedly killed after the stop phase. (Figure 16). Therefore, although the expression rate of antimicrobial peptide was low, it turned out that it shows antimicrobial activity.

Example 12 Effect of Culture Medium of Transformant Incorporated with Recombinant Vectors pPtsL-BuforinII and pPtsL-Indolicidin 1

In order to confirm the antimicrobial activity of the antimicrobial peptides, recombinant vectors pPtsL-Buforin II and pPtsL-Indolicidin prepared in Examples 8 and 9 were introduced into E. coli TOP10. After obtaining a culture medium in which the transformant was cultured, it was added to the culture medium of E. coli W3110 wild type strain and cultured in the same manner as in Example 11, followed by growth curves.

As a result, when the culture medium of the transformant expressing buforin II and indolisidine was added, E. coli compared to the control group The growth rate of W3110 appeared relatively slow (FIG. 17), which was attributed to the expressed antimicrobial peptide.

Example 13: Influence of transformant culture medium into which the recombinant vectors pPtsL-BuforinII and pPtsL-Indolicidin were introduced 2

In order to confirm the antimicrobial activity of the antimicrobial peptides, recombinant vectors pPtsL-Buforin II and pPtsL-Indolicidin prepared in Examples 8 and 9 were introduced into E. coli TOP10. After obtaining the culture medium in which the transformant was cultured, it was added to the Bacillus subtilis DB104 ( Bacillus subtilis DB104, his nprR2 nprE18 aprEΔ3 , Bacillus Genetics Stock Center, Ohio, USA) culture medium as in the method of Example 11 After incubation, growth curves were checked.

As a result, the growth rate of Bacillus subtilis DB104 was significantly slower than that of the control group when the culture medium of the transformants expressing buforin II and indolisidine was added (FIG. 18), which was expressed antimicrobial. It was found that the effect of the peptide.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram showing an expression system of a ptsL promoter capable of overexpressing a recombinant protein in the growth arrest phase of Escherichia coli.

Figure 2 shows four E. coli ( E. coli W3110, E. coli BL21 (DE3), E. coli HB101 and E. coli XL1-Blue) shows a growth curve (a: growth curve in LB liquid medium; b: growth curve in R / 2 liquid medium).

Figure 3 shows two-dimensional gel electrophoresis of E. coli W3110 (a: two-dimensional electrophoresis picture of cell lysate in exponential growth phase in LB liquid medium; b: cell lysate at growth stop in LB liquid medium; Two-dimensional electrophoresis picture; c: two-dimensional electrophoresis picture of cell lysate in exponential growth phase in R / 2 liquid medium; d: two-dimensional electrophoresis picture of cell lysate in growth stop phase in R / 2 liquid medium).

Figure 4 shows two-dimensional gel electrophoresis pictures of E. coli BL21 (DE3) (a: two-dimensional electrophoresis picture of cell lysate in exponential growth phase in LB liquid medium; b: cells at growth stop in LB liquid medium) Two-dimensional electrophoresis picture of lysate; c: Two-dimensional electrophoresis picture of cell lysate in exponential growth phase in R / 2 liquid medium; d: Two-dimensional electrophoresis picture of cell lysate solution in growth stopper in R / 2 liquid medium) .

Figure 5 shows the relative expression rate of the protein with a higher expression level in the growth stop phase than the exponential growth phase (A: E. coli W3110, the expression rate of the protein higher than the exponential growth phase in the growth stop phase; B: E. coli BL21 In the case of (DE3), the expression rate of the protein with a higher expression level than the exponential growth phase in the growth stop phase).

Figure 6 shows a genetic map of the recombinant vector pPtsL.

Figure 7 shows the genetic map of the recombinant vector pPtsL-EGFP-6His.

Figure 8 shows the growth curve and expression curve of the EGFP transformant introduced recombinant vector pPtsL-EGFP-6His.

9 is a photograph of the SDS-PAGE gel electrophoresis by concentrating the green fluorescent protein of the transformant into which the recombinant vector pPtsL-EGFP-6His was introduced (lane SM: size marker: protein standard molecular weight (97 kDa, 66 kDa, 45 kDa, Size control group representing 30 kDa, 20 kDa and 14.4 kDa); lanes 1-5: protein fraction of transformants incubated for 2 hours, 4 hours, 6 hours, 8 hours and 10 hours).

10 shows a genetic map of the recombinant vector pBAD-mlc.

Figure 11 shows the genetic map of the recombinant vector pTSLR-EGFP-6His.

Figure 12 shows the growth curve and expression curve of the transformant introduced recombinant vector pTSLR-EGFP-6His.

Figure 13 shows the genetic map of the recombinant vector pPtsL-Buforin II.

14 shows a genetic map of the recombinant vector pPtsL-Indolicidin.

Figure 15 shows the genetic map of the recombinant vector pPtsLR-Indolicidin.

16 is a transformant E. coli into which the recombinant vector pPtsL-BuforinII or pPtsL-Indolicidin is introduced. The growth curve of the TOP10.

17 is a growth curve confirming the effect of antimicrobial peptides using culture medium of transformant E. coli TOP10 into which recombinant vector pPtsL-Buforin II or pPtsL-Indolicidin was introduced to culture E. coli W3110.

FIG. 18 is a growth curve confirming the effect of antimicrobial peptides by using a culture medium of transformant E. coli TOP10 into which recombinant vector pPtsL-BuforinII or pPtsL-Indolicidin was introduced in Bacillus subtilis DB104 culture.

<110> Korea Advanced Institute of Science and Technology <120> RECOMBINANT VECTOR CONTAINING PTSL PROMOTER AND METHOD FOR          PRODUCING EXOGENEOUS PROTEINS USING THE SAME <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 1503 <212> DNA <213> Escherichia coli <400> 1 accgatgatt tcatccagtt caccgcgaca taccgggaac agactgtgcg gtgaagagag 60 cagttgctcg cggatttcat cgaccccgag attagcgtca acccagctta tttcaccgcg 120 cggcgtcatg atcccgcgca gagaacgcga cgccagcgtc agtacgccgt taatcatgta 180 acgttcttct tcggcaaatg caccttccgg gatcggcatc ggcatcgggt tatcggcatc 240 gtgctgaaca ttggcctgac gtttcccgcc catcaaacgc aggatggcat cggcagtacg 300 cgctcgcagc ggcaaagtcg actggtggcg aataaagttg cgacgcgcaa tctggttaaa 360 cacttcgatg atgatcgaga agccaatcgc ggcatacagg taacctttcg gaatgtggaa 420 accgaaacct tctgccacca gactcagacc aatcattaac aggaagctca gacagagcac 480 caccaccgtg gggtgctggt taacgaatcg cgtcagcggt ttggatgcca gcaacataac 540 cgccatcgca atcactaccg ccgccatcat caccggcaga tggttaacca tccctactgc 600 agtaattacc gcatccaacg agaagacggc gtcaaggatg acaatctgtg tgacgaccac 660 ccagaaactg gcgtagcctt taccgtggcc ggaatcatga tcgcggtttt ccagccgttc 720 atgcagttcg gttgttgctt tgaacagcaa gaatatcccc ccgaacaaca taatcaggtc 780 gcgtccggag aaggagaaat ccatgacggt aaatagcggt ttggtcagcg tgaccatcca 840 tgaaatcagc gacagcagcc ccagacgcat aatcagcgcc agtgataacc ccagcaaacg 900 cgctttatcg cgttgttttg gcggcagttt gtcagcaaga atggcgatga agaccaggtt 960 atcgataccc agcacaattt cgagaacaac aagcgtgagt agccccgccc aaattgaggg 1020 gtccattaag aattccatga caagctcctg cttaaggaat agctattcga cgccagaaat 1080 aatgcaggcg taacgacaaa atgcaaacga aaggtgcggc atagagtgcc agaaaggcag 1140 gcgttaaaag gcctgatgct gaaatgacgt cggtgacgat ccatactgcg ggctactgcc 1200 ctatactcca tggttgttaa acgggagtta aacatatcag agacgcctct gatttggcaa 1260 agatttacct tcctttgcaa acgaatgtga caaggatatt ttacctttcg aaatttctgc 1320 taatcgaaag ttaaattacg gatcttcatc acataaaata attttttcga tatctaaaat 1380 aaatcgcgaa acgcaggggt ttttggttgt agcccttatc tgaatcgatt cgattgtgga 1440 cgacgattca aaaatacatc tggcacgttg aggtgttaac gataataaga aggagatata 1500 cat 1503 <210> 2 <211> 1221 <212> DNA <213> Escherichia coli <400> 2 gtggttgctg aaaaccagcc tgggcacatt gatcaaataa agcagaccaa cgcgggcgcg 60 gtttatcgcc tgattgatca gcttggtcca gtctcgcgta tcgatctttc ccgtctggcg 120 caactggctc ctgccagtat cactaaaatt gtccgtgaga tgctcgaagc acacctggtg 180 caagagctgg aaatcaaaga agcggggaac cgtggccgtc cggcggtggg gctggtggtt 240 gaaactgaag cctggcacta tctttctctg cgcattagtc gcggggagat tttccttgct 300 ctgcgcgatc tgagcagcaa actggtggtg gaagagtcgc aggaactggc gttaaaagat 360 gacttgccat tgctggatcg tattatttcc catatcgatc agttttttat ccgccaccag 420 aaaaaacttg agcgtctaac ttcgattgcc ataaccttgc cgggaattat tgatacggaa 480 aatggtattg tacatcgcat gccgttctac gaggatgtaa aagagatgcc gctcggcgag 540 gcgctggagc agcataccgg cgttccggtt tatattcagc atgatatcag cgcatggacg 600 atggcagagg ccttgtttgg tgcctcacgc ggggcgcgcg atgtgattca ggtggttatc 660 gatcacaacg tgggggcggg cgtcattacc gatggtcatc tgctacacgc aggcagcagt 720 agtctcgtgg aaataggcca cacacaggtc gacccgtatg ggaaacgctg ttattgcggg 780 aatcacggct gcctcgaaac catcgccagc gtggacagta ttcttgagct ggcacagctg 840 cgtcttaatc aatccatgag ctcgatgtta catggacaac cgttaaccgt ggactcattg 900 tgtcaggcgg cattgcgcgg cgatctactg gcaaaagaca tcattaccgg ggtgggcgcg 960 catgtcgggc gcattcttgc catcatggtg aatttattta acccacaaaa aatactgatt 1020 ggctcaccgt taagtaaagc ggcagatatc ctcttcccgg tcatctcaga cagcatccgt 1080 cagcaggccc ttcctgcgta tagtcagcac atcagcgttg agagtactca gttttctaac 1140 cagggcacga tggcaggcgc tgcactggta aaagacgcga tgtataacgg ttctttgttg 1200 attcgtctgt tgcagggtta a 1221 <210> 3 <211> 374 <212> DNA <213> Escherichia coli <400> 3 agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt 60 ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa 120 agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga 180 ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga 240 tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct 300 aacaggagga attaaccatg aaaaaactgc tgttcgcgat tccgctggtg gtgccgttct 360 atagccatag cacc 374 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtaggcatgc accgatgatt tcatccagtt ca 32 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cccactccat atgttgctac ctcctttatt atcgt 35 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggaattcatg gtgagcaagg gcgaggag 28 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ttggatcctt agtggtggtg gtggtggtgc ttgtacagct cgtcc 45 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 catgccatgg ttgcggaaaa tcagcctg 28 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggaattctta ttaaccctgc aacagacgaa 30 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgccatgcat gcaagaaacc aattgtccat att 33 <210> 11 <211> 63 <212> DNA <213> buforin <400> 11 acccgtagca gccgtgcggg tctgcagttt ccggtgggtc gtgttcatcg tctgctgcgt 60 aaa 63 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agatatacat atgaacaata acgatctctt t 31 <210> 13 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ttggatcctt attatttacg cagcagacga tgaacacgac ccaccggaaa ctgcagaccc 60 gcacggctgc tacgggtcgc agtcgcacgt cgc 93 <210> 14 <211> 39 <212> DNA <213> indolicidin <400> 14 attctgccgt ggaaatggcc gtggtggccg tggcgtcgt 39 <210> 15 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ttggatcctt attaacgacg ccacggccac cacggccatt tccacggcag aatcgcagtc 60 gcacgtcgcg gc 72  

Claims (24)

Escherichia coli coli ) recombinant vector containing the gene encoding the ptsL promoter and foreign protein. The recombinant vector of claim 1, wherein the ptsL promoter comprises a DNA encoding a ptsL promoter activator having a nucleotide sequence of SEQ ID NO: 1. Escherichia coli coli ) -derived ptsL promoter, a recombinant vector containing a gene encoding an expression regulatory protein of ptsL gene, a promoter for expressing the gene encoding the expression regulatory protein and a gene encoding a foreign protein. The recombinant vector of claim 3, wherein the ptsL promoter comprises a DNA encoding a ptsL promoter activator having a nucleotide sequence of SEQ ID NO: 1. According to claim 3, the gene encoding the protein of the expression control ptsL gene recombinant vector, characterized in that the gene (mlc) encoding the protein, inhibiting expression of the gene in ptsL. The recombinant vector according to claim 5, wherein the mlc gene has a nucleotide sequence of SEQ ID NO. The recombinant vector according to claim 3, wherein the expression promoter of the gene encoding the expression control protein is a BAD promoter. The recombinant vector according to claim 7, wherein the BAD promoter has a nucleotide sequence of SEQ ID NO: 3. The method of claim 1, wherein the foreign protein is a peptide, polypeptide, binding protein, binding domain, adhesion protein, structural protein, regulatory protein, toxin protein, enzyme, inhibitor, hormone, hormonal analog, antibody, signal. Recombinant vector, characterized in that any one selected from the group consisting of a delivery protein, single-chain antibody, antigen, cytokine, regulatory factors and parts thereof. The recombinant vector of claim 9, wherein the peptide is an antimicrobial peptide. The recombinant vector according to claim 10, wherein the antimicrobial peptide is buforin II or indolicidin. A recombinant microorganism transformed with the recombinant vector of claim 1. The microorganism of claim 12, wherein the microorganism used for transformation is modified so as not to produce intracellular or extracellular protease related to degradation of the expressed foreign protein, in favor of intracellular overexpression of the foreign protein. Converted recombinant microorganisms. A recombinant microorganism transformed with the recombinant vector of claim 3. The trait of claim 14, wherein the microorganism used for transformation is modified so as not to produce intracellular or extracellular proteases related to the degradation of the expressed foreign protein in favor of intracellular overexpression of the foreign protein. Converted recombinant microorganisms. Culturing the recombinant microorganism of claim 12 to express the foreign protein in the growth arrestor; And recovering the expressed foreign protein. The method of claim 16, wherein the foreign protein is a peptide, polypeptide, binding protein, binding domain, adhesion protein, structural protein, regulatory protein, toxin protein, enzyme, inhibitor, hormone, hormone analog, antibody, signal transduction protein, short chain And any one selected from the group consisting of antibodies, antigens, cytokines, regulatory factors and parts thereof. 18. The method of claim 17, wherein the peptide is an antimicrobial peptide. Culturing the recombinant microorganism of claim 14 to express the foreign protein in the growth arrestor; And recovering the expressed foreign protein. The method of claim 19, wherein the foreign protein is a peptide, polypeptide, binding protein, binding domain, adhesion protein, structural protein, regulatory protein, toxin protein, enzyme, inhibitor, hormone, hormonal analog, antibody, signal transduction protein, short chain And any one selected from the group consisting of antibodies, antigens, cytokines, regulatory factors and parts thereof. The method of claim 20, wherein the peptide is an antimicrobial peptide. Method of improving foreign protein comprising the following steps: (a) constructing a variant library of genes encoding foreign proteins; (b) constructing a genetic recombinant library containing an E. coli derived ptsL promoter and a library of gene variants encoding the foreign protein such that the variant of the foreign protein is expressed by a gene encoding a ptsL promoter; (c) transforming the microbial host cell selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, actinomycetes, yeast and fungi with the genetic recombinant library or the vector comprising the genetic recombinant library; (d) culturing the transformed host cell to express the gene variant library at a cell growth arrester; And (e) screening cells expressing foreign proteins with improved traits. Method of improving foreign protein comprising the following steps: (a) constructing a variant library of genes encoding foreign proteins; (b) the E. coli-derived ptsL promoter, the gene encoding the expression regulatory protein of the ptsL gene, the promoter for expressing the gene encoding the expression regulatory protein and the foreign language so that the variant of the foreign protein is expressed by the gene encoding the ptsL promoter Preparing a genetic recombinant library containing a library of genetic variants encoding a protein; (c) transforming the microbial host cell selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, actinomycetes, yeast and fungi with the genetic recombinant library or the vector comprising the genetic recombinant library; (d) culturing the transformed host cell to express the gene variant library at a cell growth arrester; And (e) screening cells expressing foreign proteins with improved traits. The method of claim 22 or 23, wherein the screening step uses a protein that recognizes the activity of the foreign protein, a substance labeled on the foreign protein, a labeled ligand that binds to the foreign protein, or an antibody that specifically binds to the foreign protein. Method for improving foreign protein, characterized in that.

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