KR100541848B1 - A method for producing a target protein by simultaneously expressing a nonspecific DNA bindng protein Dps and a target protein, a vector using for the same and a transformed E.coli cell - Google Patents

Abstract

The present invention relates to a method for producing a target protein by simultaneously expressing a non-specific DNA binding protein dps and a target protein in Escherichia coli, specifically, (a) a recombinant gene encoding a non-specific DNA binding protein dps of E. coli and purpose culturing the recombinant Escherichia coli (E.coli) containing a recombinant gene encoding a protein and (b) for the production method of a target protein characterized in that it comprises the step of separating the desired protein from the culture will be. The present invention also relates to an expression vector used in the method and E. coli transformed with the vector.

Nonspecific DNA Binding Proteins, Dps, Target Proteins

Description

Method for producing a target protein by simultaneously expressing a nonspecific DNA bindng protein Dps and a target by simultaneously expressing nonspecific DNA binding protein dps and target protein in E. coli protein, a vector using for the same and a transformed E.coli cell}

1 is a diagram illustrating a process of constructing a pYS01 vector including a Dps gene.

FIG. 2 is a diagram illustrating a process of constructing a pYS02 vector including a gene encoding a fusion protein of a Dps gene, a polymorphic protein (Polh), and a green fluorescent protein (GFP).

3 is a graph showing the cell growth curve. Where (A) and (B) are cultured in M9 minimal medium and LB complex medium, respectively, and (○) and (●) are experimental groups co-expressing control BL21 [pMPL102] and Dps without co-expressing Dps, respectively. BL21 [pYS02].

Figure 4 is a control group BL21 [pMPL102] not co-expressing Dps and experimental group BL21 [pYS02] co-expressing the Dps of the present invention to the target protein Polh / GFP of whole cells over time after IPTG induction This is a result of comparing the expression patterns through SDS-PAGE analysis at the same cell concentration. Here, (A) and (B) show the results when cultured in M9 minimal medium and LB complex medium, respectively.

Lane 1 (M): protein molecular weight size marker

2-lane (0-): whole cell protein profile obtained immediately after induction (0 h) in expression vector pMPL102

Three-lane (0+): whole cell protein profile obtained immediately after induction (0 hours) in expression vector pYS02

Four-lane (1-): whole cell protein profile obtained 1 hour after induction by expression vector pMPL102

Lane 5 (1+): Whole cell protein pattern obtained 1 hour after induction from expression vector pYS02.

Lane 6- (2-): Whole cell protein pattern obtained 2 hours after induction of expression vector pMPL102

Lane 7 (2+): Whole cell protein pattern obtained 2 hours after induction of expression vector pYS02.

Eight lanes (3-): Whole cell protein profiles obtained 3 hours after induction with the expression vector pMPL102.

Lane 9 (3+): Whole cell protein profile obtained 3 hours after induction with expression vector pYS02.

Lane 10 (4-): Whole cell protein profile obtained 4 hours after induction with the expression vector pMPL102.

Lane 11 (4+): Whole cell protein profile obtained 4 hours after induction with expression vector pYS02.

Lane 12 (6-): Whole cell protein profile obtained 6 hours after induction with expression vector pMPL102

Lane 13 (6+): Whole cell protein profile obtained 6 hours after induction with expression vector pYS02

FIG. 5 shows Western blotting of poly-histidine peptide antibody after induction of IPTG by incubating control BL21 [pMPL102] without co-expressing Dps and experimental group BL21 [pYS02] co-expressing Dps of the present invention in LB medium. It is a view visualizing the expression level of the target protein Polh / GFP through.

Lane 1- (1): The target protein of the sample obtained 1 hour after induction by the expression vector pMPL102.

2-lane (1+): target protein of sample obtained 1 hour after induction from expression vector pYS02

3-lane (2-): target protein of sample obtained 2 hours after induction by expression vector pMPL102

Four-lane (2+): target protein of sample obtained 2 hours after induction with expression vector pYS02

5-lane (4-): target protein of sample obtained 4 hours after induction by expression vector pMPL102

6 lane (4+): target protein of sample obtained 4 hours after induction with expression vector pYS02

7 lane (6-): target protein of the sample obtained 6 hours after induction with the expression vector pMPL102.

Eight lanes (6+): protein of interest obtained 6 hours after induction with expression vector pYS02

Figure 6 is a graph showing the ratio of the target protein of the total protein of all cells over time. Where (A) and (B) are cultured in M9 minimal medium and LB complex medium, respectively, and (○) and (●) are experimental groups co-expressing control BL21 [pMPL102] and Dps without co-expressing Dps, respectively. BL21 [pYS02].

Figure 7 is a graph comparing the expression of the target protein with the passage of time for the same cell concentration after expression induction. Where (A) and (B) are cultured in M9 minimal medium and LB complex medium, respectively, and (○) and (●) are experimental groups co-expressing control BL21 [pMPL102] and Dps without co-expressing Dps, respectively. BL21 [pYS02].

How the present invention is cultured to produce the desired protein a vector, the transformed E. coli containing them, and the E. coli capable of expressing a non-specific DNA binding protein dps and the desired protein of Escherichia coli (E.coli) simultaneously in E. coli It is about.

In the meantime, many researches have been conducted to increase the yield of genetic engineering technology for producing recombinant proteins in organisms such as microorganisms, yeast, insect cells and the like. In particular, because E. coli has a relatively small number of genes compared to other biological systems, the metabolic process of gene analysis and protein expression has been revealed in detail. Therefore, studies have been actively conducted on a system using Escherichia coli as a production strain for the case where the recombinant protein does not require post-translational modification, and Escherichia coli which can obtain a higher yield in recombinant protein production. Many approaches have been devised because of the multiple approaches to developing production strains.

However, conventionally studied methods usually use strong promoters or enhance transcription by binding to promoters such as factor for inversion stimulation (Fis) and modifying gene sequences to enhance their efficiency in translation. Approach in the same direction. These approaches are limited to manipulating vectors independently of changes in the cell's growth environment, and that various environmental changes and stresses that may occur during culturing adversely affect recombinant protein production by turning the E. coli metabolic activity. I overlook things.

In general, as the cell concentration increases during the cultivation of microorganisms including E. coli, the environmental conditions around the cells change with time, and when this change significantly affects the proliferation and metabolic activity of cells, this yields a yield in producing recombinant proteins. It is a large cause of deterioration. For example, oxygen radicals, which are part of the metabolism of Escherichia coli, are very small, but can cause significant damage to cellular activity by damaging not only organelles but also DNA (D. McDougald et al., 2002, Antonie Van Leeuwenhoek 81, 3-13; A. Martinez et al., 1997, J. Bacteriol. 179, 5188-5194; SH Choi et al. , 2000, Appl. Environ.Microbiol . 66, 3911-3916), with acetic acid in high concentration cultures. In addition to the accumulation of the same secondary metabolites, depletion of nutrients such as carbon sources and amino acids can have a fatal effect on normal metabolic and production activities (A. Martin, 1991, Mol. Microbiol. 5, 3-10; AC St John et al., 1980, J. Bacteriol. 143, 1223-1233). In addition, overexpression of exogenous proteins in Escherichia coli has been reported to induce the expression of heat shock proteins, thereby affecting the activity of the cells, and causing a decrease in yield by partially degrading the protein of interest. DB Archer et al., 1992, Biotechnol. Lett. 14, 357-362; Y. Jubete et al. , 1996, J. Biol. Chem. 217, 30798-30803; SW Harcum et al. , 1993, Biotechnol. Bioeng. 42, 675-685 DM Ramirez et al., 1995, Biotechnol. Bioeng. 47, 596-608).

Thus, cells cope with environmental changes by promoting the expression of universal proteins that can cope with specific or multiple stresses in order to cope with these deadly stresses. One such protein is DNA-binding protein from starved cells (dps), a nonspecific DNA binding protein, which contains about seven nonspecific DNA binding proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) in Escherichia coli. One of them is not only when products that induce oxidative action in cells such as hydrogen peroxide and free radicals are produced or added, but also mainly in an environment depleted of nutrients such as glucose and amino acids, such as the stationary phase of cell growth. It is a protein that increases in amount and is a protein that responds to damage caused by oxidation and growth stresses (G. Zhao et al. , 2002, J. Biol. Chem. 277, 27689-27696; A. Almiron et al. , 1992, Genes Dev. 6, 2646-2654; A. Ishihama, 1999, Genes to Cells 4, 135-143; T. Ali Azam et al., 1999, J. Biotechnol. 181, 6361-6370).

Therefore, the present inventors, when producing recombinant protein in Escherichia coli, is not a one-dimensional approach to increase the level of transcription and translation irrespective of the surrounding environmental conditions, but the various types of cell growth during overexpression of the recombinant protein When co-expressing Dps, which not only copes with inhibitors, but also helps in the expression of the protein of interest, the present inventors have found that the yield of the protein of interest has been improved by more than two times.

An object of the present invention is to provide a vector capable of simultaneously expressing Dps and a target protein in Escherichia coli and E. coli transformed with the vector.

It is also an object of the present invention to provide a method for producing a high yield of a target protein using E. coli, which can include and express Dps and a target protein gene.

The present invention provides an E. coli expression vector comprising a recombinant gene encoding a non-specific DNA binding protein dps of E. coli and a recombinant gene encoding a protein of interest. Preferably, the expression vector is pYS02.

The present invention also provides Escherichia coli (E.coli) non-specific DNA binding protein of Escherichia coli (E.coli) transformed with the E. coli expression vector containing the recombinant gene and the purpose of recombinant gene encoding a protein encoding the same time of dps . Preferably, the E. coli is E. coli (KCTC 10426BP).

The invention further culturing the (a) E. coli, the recombinant Escherichia coli (E.coli) containing a recombinant gene encoding a recombinant gene and the protein of interest encoding a non-specific DNA binding protein of dps (E.coli) and

(b) providing a method for producing a target protein comprising the step of separating the target protein from the culture. The nonspecific DNA binding protein is preferably a nonspecific DNA binding protein of E. coli K-12. In addition, the recombinant E. coli ( E. coli ) is preferably E. coli (KCTC 10426BP). In addition, the separation of the desired protein may be used conventional protein separation methods such as salting out, ion chromatography, affinity chromatography and filtration.

In the present invention, "E. coli non-specific DNA-binding protein dps" is derived from E. coli, nutrients such as glucose and amino acids, if there is a product that causes intracellular oxidation, such as hydrogen peroxide and free radicals It is a protein that increases the amount of expression in this depleted environment, and refers to a protein corresponding to various stresses of oxidative damage and growth arrest.

In one embodiment of the present invention, an expression vector was prepared to co-express Dps from E. coli K-12, which is known to be expressed under various stress conditions, including cell growth arrester, and E. coli. For this purpose, PCR-amplified Dps genes were inserted after recombinant target protein genes controlled by one promoter and having a ribosomal binding site (RBS) using molecular biological techniques. The amplified insert was designed to additionally have its own RBS in front of the Dps. Therefore, when inducing expression, one mRNA corresponding to a region from the gene of the target protein to the gene corresponding to the Dps is transcribed, but since each has its own RBS, the Dps is designed to co-express with the target protein. Thus, by inserting the co-expression of the Dps as described above, it overcomes the yield reduction phenomenon that occurs when overexpressing the recombinant protein, and provides an E. coli expression system that can improve the production of the recombinant protein regardless of the medium composition.

In one embodiment of the present invention, the expression vector pYS02 for E. coli, including the Dps gene derived from E. coli K-12, was deposited with No. 10426BP dated February 18, 2003, to the Korea Biotechnology Research Institute Gene Bank (KCTC).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1. Preparation of recombinant vector for co-expression of Dps and target protein

PCR (polymerase) using a chromosome of E. coli K-12 as a template and primers Dps01 (upstream) (SEQ ID NO: 1 with Nhe I recognition site) and Dps02 (downstream) (SEQ ID NO: 2: EcoR I recognition site) Through the chain reaction, the entire gene of Dps (NCBI genebank accession number: x69337) was massively amplified. Next, the amplified Dps gene was inserted into the E. coli expression vector pTrcHisC (Invitrogen, USA) using Nhe I and EcoR I restriction enzyme sites. The recombinant vector thus produced was named pYS01 (4821 bps). 1 is a diagram illustrating a process of manufacturing a pYSO1 vector. PCR using the Dps gene (SEQ ID NO: 3) containing the RBS portion of the pYS01 and a poly-histidine tag as the primers using Dps03 (upstream) (SEQ ID NO: 4) and Dps02 (downstream) (SEQ ID NO: 2) amplification, and the desired protein of the Polyhedrin protein through the (polyhedrin; Polh) gene and the green fluorescent protein; Pst on (green fluorescent protein GFP) vector pMPL102 (KCTC Accession No. 10243BP call) including the fusion protein gene the gene fusion I and EcoR I restriction sites were inserted. The resulting vector was named pYS02 (6419 bps) and is a vector designed to co-express Dps with a foreign protein of interest in the present invention. 2 is a diagram illustrating a process of manufacturing the pYS02. Polh / GFP, a protein of interest, has the characteristic of being formed as an inclusion body when expressed in Escherichia coli (see Cha Hyung-jun, Seo Jung-hyun, Lyrin, Korean Patent Application No. 2002-0036063).

Each PCR reaction was denatured at 99 ° C. for 5 minutes under conventional reaction conditions (10 mM pH9.0 Tris-HCl, 50 mM KCl, 0.1% Triton X-100, 2 mM MgSO ₄ ), followed by Taq DNA polymerase (Promega, USA). After the addition, all were repeated 30 times at 95 ℃ / 1 minute (denatured), 55 ℃ / 30 seconds (restoration), 72 ℃ / 40 seconds (extension). After PCR, the amplified DNA was identified and purified on 1% agarose gel and used for restriction enzyme reaction. Restriction enzyme-treated DNA fragments were inserted into the vectors pTrcHisC and pMPL102, respectively, as shown in FIGS. In order to confirm the insertion of the inserted gene at each step, the plasmid was extracted from the transformed Escherichia coli and treated with the restriction enzyme at the original insertion site to electrophores the size of the cut DNA fragment on 1% agarose gel. Used.

Example 2. Preparation of E. coli transformants

In the method of transforming Escherichia coli Top10 (Invitrogen, USA), which is commonly used for cloning, and BL21, which is used for expression, a heat shock (42 ° C.) was made after making competent cells using CaCl ₂ buffer. The plasmid was introduced into the host cell by the method. Transformed colonies were selected using antibiotic ampicillin due to the nature of the plasmids used.

Example 3 Culture of Escherichia Coli and Co-Expression of Recombinant Protein and Dps

Each of the bacteria that were confirmed for the accuracy of transformation was cultured in LB medium for about one day (20 hours) after glycerol (glycerol) concentration to 15%, and then stored at -80 ℃ until culture experiments. Cultivate 200 μl of the above storage bacteria solution with 5 ml of M9 medium (12.8 mg ml ^-1 Na ₂ HPO ₄ ㆍ 7H ₂ O, 3 mg ml ^-1 KH ₂ PO ₄ , 0.5 mg ml ^-1 NaCl, 1 mg ml ^-1) Ampicillin in NH ₄ Cl, 3 mg ml ^-1 CaCl ₂ , glucose 0.5%) or LB complex medium (5 mg ml ^-1 yeast extract, 10 mg ml ^-1 Tryptone, 10 mg ml ^-1 NaCl) (50 μg / ml) and incubated for 12 hours, and then the culture was inoculated into a 250 ml flask containing 60 ml of the same medium. At this time, the inoculation was made to a final cell concentration of 0.2. The culture was performed at 37 ° C. and 250 rpm. When the absorbance of the culture solution at 600 nm wavelength was about 1.0 for protein expression, the expression of recombinant target protein was induced by adding IPTG (final 1 mM), which is an inducer, isopropylthio-β-D-galactoside (IPTG).

Example 4. Measurement and Quantitation of Recombinant Protein Expression

Differences in recombinant protein expression according to co-expression of Dps were compared and analyzed by two methods, SDS-PAGE and Western blotting. To this end, samples were taken from the culture medium every hour to measure optical density (OD) (FIG. 3). In addition, after centrifugation of 200μl sample was removed the supernatant cell precipitate was stored at -80 ℃ was used for analysis. For SDS-PAGE analysis, dilute it in the sample buffer (0.5M Tris-HCl, pH 6.8; 10% glycerol; 5% SDS; 5% β-mercaptoethanol, and 0.25% bromophenol blue) and boil at 100 ° C for 5 minutes. Denatured. The amount of buffer used was such that the OD of the sample was five. After electrophoresis on 15% SDS-polyacrylamide gel, the gel was stained with Coomassie blue (Bio-Rad, USA) solution, and image analysis software (Gel-Pro; Media Cybernetics, USA) was used. Analyzes through (Fig. 4).

In the case of western blotting, electrophoresis was completed on 15% SDS-polyacrylamide gel in the same manner as in SDS-PAGE analysis, and then transferred to a nitrocellulose membrane under a voltage of 15V. Synchronization of Dps by Detecting and Coloring Recombinant Protein Transfected Nitrocellulose Membrane by Detecting and Coloring Recombinant Protein and Dps with Poly-Histidine Label Using Monoclonal Antibody to Poly-Histidine Peptide (Santa Cruz Biotechnology, USA) The amount of expression of the target protein according to the expression was quantified (Fig. 5).

When the recombinant Dps is co-expressed in Example 4, the expression level of the recombinant protein Polh / GFP is compared with the control group in which the recombinant Dps is not expressed. The increase was visualized (Figs. 4 and 5), and when co-expression of recombinant Dps through quantitative analysis of Fig. 4, it was confirmed that the high yield improvement of the target protein regardless of the medium (Figs. 6 and 7). .

In addition, as a result of comparing the production yield of the target protein under the co-expression of recombinant Dps according to the medium condition at the time when the difference of these results is the maximum, the production yield for the same cell concentration in the M9 minimum medium was relatively high as 2.06. The production yield for the same volume of culture was 1.63 (Table 1). On the other hand, in the case of LB complex medium, the production yield for the same cell concentration was 2.10, which is similar to that of M9 medium, whereas the production yield for the same volume of culture medium was 2.45, which was significantly increased compared to M9. This is because, in the M9 minimal medium, when the Dps is co-expressed, there is a decrease in cell growth, whereas the LB complex medium maintains a relatively high cell concentration compared to the control group. Therefore, it can be seen that when the culture in a certain volume of the incubator using the LB complex medium can bring a higher yield.

Table 1. Increase in the maximum yield of the target protein relative to the control

division M9 Minimum Badge LB Composite Medium Increase rate of production yield per same cell concentration 2.06 2.10 Increase rate of production yield per volume 1.63 2.45

From the results of the above examples, it was found that when the Dps is expressed simultaneously with the target protein, the production yield of the target protein can be greatly increased. In addition, the medium used at this time was found that the composite medium is more efficient than the minimum medium. E. coli expression vector pYS02 containing the Dps gene derived from E. coli K-12 obtained in the above example was deposited in the Korea Biotechnology Research Institute Gene Bank (KCTC) February 18, 2003 Accession No. 10426BP.

The expression vector of the present invention and transformed E. coli comprising the same can be used to express Dps at the same time as the target protein to produce the target protein in high yield.

According to the method for producing a target protein of the present invention, by simultaneously expressing the target protein in Dps and cells, the yield of the target protein can be greatly increased.

<110> Pohang University of Science and Technology <120> A method for producing a target protein by simultaneously          expressing anonspecific DNA bindng protein and a target protein,          a vector using for the same and a transformed E.coli cell <130> PU000305 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for DPS gene amplification: NheI restriction site <400> 1 ggggctagca tgagtaccgc taaattagt 29 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for DPS amplification: EcoRI restriction site <400> 2 gggaattctt attcgatgtt agactc 26 <210> 3 <211> 650 <212> DNA <213> Artificial Sequence <220> <223> RBS-poylhistidine-DPS recombinant gene <400> 3 ctgcaggtgg gcactcgacc ggaattatcg attaacttta ttattaaaaa ttaaagaggt 60 atatattaat gtatcgatta aataaggagg aataaaccat ggggggttct catcatcatc 120 atcatcatgg tatggctagc atgagtaccg ctaaattagt taaatcaaaa gcgaccaatc 180 tgctttatac ccgcaacgat gtctccgaca gcgagaaaaa agcaacagta gagttgctga 240 atcgccaggt tatccagttt attgatcttt ctttgattac caaacaagcg cactggaaca 300 tgcgcggcgc taacttcatt gccgtacatg aaatgctgga tggcttccgc accgcactga 360 tcgatcatct ggataccatg gcagaacgtg cagtgcagct gggcggtgta gctctgggga 420 ccactcaagt tatcaacagc aaaaccccgc tgaaaagtta cccgctggac atccacaacg 480 ttcaggatca cctgaaagaa ctggctgacc gttacgcaat cgtcgctaat gacgtacgca 540 aagcgattgg cgaagcgaaa gatgacgaca ccgcagatat cctgaccgcc gcgtctcgcg 600 acctggataa attcctgtgg tttatcgagt ctaacatcga ataagaattc 650 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer for RBS-polyhistidine-DPS amplification: PstI          restriction site <400> 4 tactgcaggt gggcactcga ccggaattat cg 32

Claims (9)

 Escherichia coli expression vector pYS02 comprising a recombinant gene encoding a non-specific DNA binding protein dps (DNA-binding protein from starved cells) of E. coli of SEQ ID NO: 3 and a recombinant gene encoding a target protein. delete The E. coli expression vector according to claim 1, wherein the target protein is a foreign protein. E. coli transformed with the expression vector according to claim 1. The transformed Escherichia coli ( E. coli ) according to claim 4, characterized in that E. coli (KCTC 10426BP). (a) The recombinant E. coli (E. coli) comprising a recombinant gene encoding a non-specific DNA binding protein dps (DNA-binding protein from starved cells) of E. coli and a recombinant gene encoding a target protein. coli) and (b) separating the target protein from the culture. The method of claim 6, wherein the non-specific DNA binding protein dps (DNA-binding protein from starved cells) is a non-specific DNA binding protein dps of E. coli K-12. . The method of claim 6, wherein the recombinant E. coli is E. coli (KCTC 10426BP). The method of claim 6, wherein the target protein is a foreign protein.

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