KR20090025481A - Preparation method of recombinant protein by use of rpos as a fusion expression partner - Google Patents

Abstract

A method for manufacturing a recombinant protein using RpoS(RNA polymerase sigma factor) is provided to improve yield of the recombinant protein due to a small size of the RpoS. A method for manufacturing a recombinant protein using RpoS(RNA polymerase sigma factor) comprises the following steps of: manufacturing an expression vector by connecting a heterologous protein to a RpoS gene as a fusion partner; manufacturing a transformant by employing the expression vector to a host cell; and inducing expression of the recombinant protein by cultivating the transformant.

Description

Production method of recombinant protein using RSP as a fusion partner {PREPARATION METHOD OF RECOMBINANT PROTEIN BY USE OF RpoS AS A FUSION EXPRESSION PARTNER}

The present invention relates to an expression vector of a recombinant protein using RpoS (RNA polymerase sigma factor) as a fusion partner, and a method for producing a recombinant protein using the same, and more particularly, to connect a fusion partner and a gene of a foreign protein. A method of producing a recombinant protein comprising the steps of preparing an expression vector, introducing the expression vector into a host cell to produce a transformant, and culturing the transformant to induce expression of and obtain the recombinant protein. The present invention relates to a method for producing a recombinant protein, characterized in that to improve the water solubility and folding (folding) of the recombinant protein by using the expression of the RpoS gene as a fusion partner, and to an expression vector containing the gene of RpoS as a fusion partner.

Currently, proteins produced by biotechnology can be roughly divided into pharmaceutical and research proteins such as immunomodulatory and enzyme inhibitors and hormones, and industrial proteins such as diagnostic proteins and reactive enzymes. Technology development and industrialization are being promoted. In particular, when producing useful recombinant proteins using recombinant microbial technology, E. coli is well known for its genetic information, has a variety of vector systems, and has the advantage of being able to cultivate rapidly at high concentrations in relatively inexpensive media. It is used.

In producing recombinant proteins in Escherichia coli, various expression vectors with powerful inducible promoters have been developed and used for the production of foreign proteins. However, when E. coli is used as a host cell, the protein to be produced is often degraded by proteolytic enzymes in E. coli, and the yield is low. In particular, the expression of a small polypeptide having a molecular weight of 10 kDa or less is known to be severe. have. In addition, recombinant proteins often form insoluble aggregates, known as inclusion bodies, when they are overexpressed in Escherichia coli. In the case of polypeptides expressed as aggregates, the folding intermediates are intermolecular disulfides. Inferior purity in aggregates of the desired polypeptide by non-selective binding to other protein impurities (chaperon, ribosome, early factor, etc.) of the host cell by bond or hydrophobic interaction There are disadvantages. In addition, in order to make the expressed protein active, it must be refolded by dissolving and diluting with a denaturant such as guanidine hychloride or urea. Production yields are reduced (Marston FA, Biochem J 240 (1): 1-12, 1986).

In order to obtain high yield of active recombinant protein in Escherichia coli, the expression rate of Escherichia coli is lowered to increase the acceptability of the target protein, thereby lowering temperature expression (Hammarstrom et al. , Protein Sci . 11: 313-321, 2002), using various promoters or optimizing induction conditions (Qing et al . , Nat Biotechnol . 22: 877-882, 2004) and co-expression of molecular chaperone or protein folding regulators with protein of interest (de Marco & De Marco, J Biotechnol . 109: 45-52, 2004). Fusion of the partner and the protein of interest is the most common method.

Indeed, several fusion partners have been studied and reported over the past several years that induce high water-soluble expression of foreign proteins in Escherichia coli (Esposito & Chatterjee, Curr Opin). Biotechnol . 17: 353-358 2006; Kapust & Waugh, Protein Sci . 8: 1668-1674, 1999; Sachdev & Chirgwin, Biochem . Biophys . Res . Commun . 244: 933-937, 1998). Numerous fusion partners have been developed and used, but the most studied fusion partners are maltose binding protein (MBP), glutathione-S-transferase (GST) and thioredoxin ( Thioredoxin (Trx), NusA, and the like. In the case of maltose-binding protein, a large hydrophobic gap, in which hydrophobic amino acids are gathered in the part involved in dimer formation, effectively hides the hydrophobic portion of the newly synthesized protein, thereby preventing the target protein from becoming an insoluble aggregate. Toxins help disulfide binding of the protein of interest, and NusA has a very good ability to fold into the active form when overexpressed in Escherichia coli, leading to the correct folding of the protein of interest (Bach H et. al . , J Mol Biol 312: 79-93, 2001; Edward RL et al . , Nat Biotechnol 11: 187-193, 1993; Davis GD et al . , Biotechnol Bioeng 65: 382-388, 1999)

Such a fusion partner has the advantage of easily purifying the target protein expressed by affinity chromatography and is known to help fold the target protein using different molecular biological properties. However, these fusion partners have a disadvantage in that the yield of the target protein is considerably decreased according to the size of the fusion moiety, since it is relatively large compared to the target protein, and it does not have a general purpose for both the medical and industrially useful proteins. In addition, even when inducing the expression of the target protein in water-soluble, it often fails to induce the expression of the active form that performs its own function, and in the process of removing the fusion partner in order to be used for proper use There is a problem of irrationality.

Until now, domestic recombinant protein produced industrially by biotechnology has been focused on the development of the production process of expressible recombinant proteins, so the development of expression system, which is a core source technology, is developed as an additional technology of imitation or improvement level compared to other countries. The proteins that are present and are of great commercial and medicinal importance are secretory and membrane proteins, which are difficult-to-express proteins that produce insoluble aggregates when expressed in Escherichia coli. It is becoming. In order to overcome this, it is important to secure a source-based technology for the expression system of E. coli protein in E. coli.

In addition, as described above, since each fusion partner has different molecular biological characteristics, the fusion recombinant protein may have different mechanisms to assist folding, so that not all proteins have the same effect. In order to build an expression system using a fusion partner that helps to fold a recombinant protein more universally, it is necessary to find a fusion partner from a different point of view and build a new concept of a fusion partner library from a different point of view.

Recombinant protein overexpression in Escherichia coli has been known in previous reports to have a similar effect on stress from the external environment (heat shock, amino acid depletion, etc.) (Hoffman F & Rinas U, Adv). Biochem Eng Biotechnol , 89: 73-92, 2004). In view of the above report, the present inventors have attempted to develop a new concept of fusion partner. As a result of stress that inhibits the correct folding of E. coli and the overexpressed protein as a fusion partner, various target proteins were produced by genetic recombination technology, resulting in a large increase in water soluble expression for various target proteins and changes in enzyme activity. The present invention has been completed by confirming that the production of recombinant protein is possible.

An object of the present invention is to produce a protein of interest in the form of an insoluble aggregate from a transforming microorganism, by using a universal fusion partner that can be used in various target proteins while separating the insoluble aggregate into active proteins without using denaturing or reducing agents. It is to provide a method for producing a recombinant protein.

In order to achieve the above object, the present invention comprises the steps of: 1) preparing an expression vector connecting the gene of RpoS (RNA polymerase sigma factor) and the gene of the foreign protein as a fusion partner;

2) preparing a transformant by introducing the expression vector into a host cell; And,

3) culturing the transformant to induce the expression of the recombinant protein and to provide a method for producing a recombinant protein consisting of obtaining the same.

In addition, the present invention provides an expression vector, characterized in that the fusion partner is a gene of RpoS in the expression vector that is linked to the gene of the foreign protein is linked to produce a recombinant protein.

The present invention also provides a transformant transformed with the expression vector of the present invention.

In addition, there is provided a recombinant fusion protein produced by the method of the present invention.

Hereinafter, the term used by this invention is demonstrated.

"Heterologous protein or target protein" is a protein that a person skilled in the art intends to produce in large quantities, and inserts a polynucleotide encoding the protein into a recombinant expression vector to express all proteins that can be expressed in a transformant. it means.

"Recombinant protein or fusion protein" refers to a protein in which another protein is linked to or added to the N-terminus or C-terminus of the sequence of the original foreign protein.

An "expression vector" is a linear or circular DNA molecule consisting of fragments encoding a polypeptide of interest operably linked to additional fragments provided for transcription of the expression vector. Such additional fragments include promoter and termination code sequences. Expression vectors also include one or more replication initiation points, one or more selection markers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both.

Hereinafter, the present invention will be described in detail.

The present invention 1) preparing an expression vector connecting the gene of RpoS and the gene of the foreign protein as a fusion partner (fusion partner);

2) preparing a transformant by introducing the expression vector into a host cell; And,

3) culturing the transformant to induce the expression of the recombinant protein and to provide a method for producing a recombinant protein consisting of obtaining the same.

In Escherichia coli, foreign proteins are often expressed as insoluble aggregates. Therefore, the present invention was intended to improve the water-soluble expression of recombinant proteins using RpoS, an E. coli protein whose expression increases when given stress that inhibits correct folding of the protein, as a fusion partner. The protein remains active even under conditions that inhibit the folding of the protein, and the increased expression level indicates that the protein structure is stable and has excellent self-folding ability as well as the possibility of being involved in the biomechanism of E. coli to overcome the misfolding of the protein. As shown, it can be effectively used as a fusion protein.

Specifically, E. coli protein with increased expression when GdnHCl, which shows a proteolytic effect, was given stress to inhibit correct folding of the protein was selected (see FIG. 1). As a result of analyzing and identifying the protein, E. coli RpoS ( It was confirmed that the expression level of RNA polymerase sigma factor was increased (see Table 1). When a protein having increased water-soluble expression against proteolytic stress is used as a fusion partner, it will maintain high water-soluble expression of the protein of interest. When GdnHCl was added to the medium to maintain the growth of E. coli, two-dimensional electrophoresis was performed to compare the changes in the protein body of E. coli. It was confirmed (see FIG. 1). Proteins with increased expression levels were analyzed and identified using Matrix-Assisted Laser Desorption / Ionization-Time Of Flight (MALDI-TOF) -MS to confirm that they were RpoS (see Table 1).

The foreign protein may be any protein desired by those skilled in the art, and in particular, a biological activity selected from the group consisting of medical, research and industrial proteins such as antigens, antibodies, cell receptors, enzymes, structural proteins, serum, and cellular proteins. Various foreign proteins with can be expressed as recombinant proteins. In the present invention, RpoS was fused and expressed with 10 proteins known to form insoluble aggregates when expressed alone without a fusion partner in E. coli to verify the potential as a fusion partner to help fold foreign proteins. That is, medical and research proteins typically include human minipro-insulin (MP-INS), human epidermal growth factor (EGF), human prepro-grelin (human prepro-). ghrelin (ppGRN), human interleukin-2 (hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase, hereinafter GAD _{448 -585),} also the Pseudomonas footage (Pseudomonas putida ) cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF) and cold Cold autoinflammatory syndrome (NALP3) Nacht domain (hereinafter referred to as Nacht) was fusion-expressed as the target protein, and industrial proteins were 448 to 558 amino acids of glutamic acid decarboxylase known as diagnostic markers of type 1 diabetes. glutamate decarboxylase _448-585, hereinafter 'GAD _448-585') and is used in the pretreatment of fiber may also receiving attention as environmentally friendly industrial enzyme brought plastic resolution footage is Pseudomonas (Pseudomonas putida ) -derived cutinase (hereinafter referred to as 'CUT') was selected as the target protein, respectively, to prepare expression vectors inserted into the carboxyl ends of RpoS (see FIG. 2), and then transformed into E. coli and recombinant proteins therefrom. Produced in the form. As a result of confirming the expression level of each recombinant protein, it was confirmed that the expression of the foreign protein fused with RpoS was more in the water-soluble expression than the insoluble aggregate (see FIG. 3), and also the RpoS than the water-soluble expression of the foreign protein expressed alone. It was confirmed that the amount of water-soluble expression in the foreign protein fused with and very increased (see Fig. 4).

In addition, PNP specific degradation activity of recombinant cutinase (RpoS :: CUT), which is an industrial protein that can be used without separately removing a fusion expression partner, was verified to verify the activity of the recombinant protein by the above method (see FIG. 5). ).

In addition, the present invention provides an expression vector, characterized in that the fusion partner is a gene of RpoS in the expression vector that is linked to the gene of the foreign protein is linked to produce a recombinant protein.

The RpoS gene and the foreign protein gene are linked to the skeletal vector as a fusion partner, and the skeletal vector which can be used in the method of the present invention is not particularly limited, but is not limited to pT7, pET / Rb, pGEX, pET28a, pET- Various vectors capable of transforming E. coli selected from the group consisting of 22b (+) and pGEX can be used. The expression vector of the present invention can express the fusion partner and the foreign protein as a recombinant protein by including a sequence of insertion of the gene of the fusion partner and the gene of the foreign protein in the pT7 skeleton vector in a sequence (see FIG. 2). ).

Furthermore, a polynucleotide encoding a protein cleavage enzyme recognition site may be linked between the RpoS gene and the foreign protein to be operable to the gene of the fusion partner of the vector of the present invention. When the target protein to be fused with the fusion partner is an industrial protein, the fusion partner may decrease the function of the target protein, and when the protein is a medical protein, the fusion partner may be removed because it may cause an antigen-antibody reaction. In this case, the protein cleavage enzyme recognition site may be used as the Xa factor recognition site, the enterokinase recognition site, the Genenase I recognition site, or the Furin recognition site, or any two or more of them are sequentially connected. have. In an embodiment of the invention, the vector of the present invention recombines the fusion partner and the foreign protein by including a sequence of insertion of the gene of the fusion partner, the protein cleavage enzyme recognition site, and the foreign protein gene into the pET28a skeleton vector. Can be expressed as a protein.

In an embodiment of the invention, the foreign gene can be cloned through a restriction enzyme site, and if a polynucleotide encoding a protein cleavage site is used, it is linked in frame with the polynucleotide, When secreted by a protein cleavage enzyme after secreting the foreign protein, the original protein can be produced.

In addition, a polynucleotide encoding a tag for separation and purification may be linked to facilitate separation and purification of a recombinant protein to be operable to a fusion partner or a foreign protein gene of the vector of the present invention. At this time, the separation and purification tag may be used alone, or GST, poly-Arg, FLAG, poly-His and c-myc, or any two or more of them in sequence. In an embodiment of the present invention, the vector of the present invention comprises the fusion partner, the foreign protein, and the separation and purification of the fusion partner by inserting the gene of the fusion partner, the foreign protein gene, and the site for inserting the separation and purification tag in a sequence. It can be expressed as a recombinant protein containing a dragon tag. In a preferred embodiment of the present invention, the polynucleotide encoding the poly-His tag is linked, and after the protein expression and purification, the recombinant protein is purified using a poly-His tag, and only the recombinant protein is purified using SDS-PAGE. It was confirmed that (see Fig. 6-1), HPLC analysis confirmed that the presence of a single peak (peak) (see Fig. 6-2). As the protein forms the same tertiary structure, the HPLC analysis results in a single peak, which means that the recombinant protein of the present invention is structurally constant and simultaneously exhibits the same activity.

In another aspect, the present invention provides a transformant in which the expression vector is transfected, characterized in that the fusion partner and the gene of the foreign protein is linked to produce a recombinant protein, wherein the fusion partner is a gene of RpoS. .

In addition, the present invention 1) preparing an expression vector connecting the gene of RpoS and the gene of the foreign protein as a fusion partner (fusion partner);

2) preparing a transformant by introducing the expression vector into a host cell; And,

3) It provides a recombinant fusion protein produced by the method for producing a recombinant protein consisting of culturing the transformant to induce the expression of the recombinant protein and to obtain it.

The fusion partner RpoS of the recombinant protein of the present invention is small in size, so that the yield of the target protein can be much improved compared to the previous fusion partner. In addition, as shown in Figures 3 to 4 can act universally to all of the medical or industrially useful proteins, as shown in Figure 5 not only induces water-soluble expression of the protein of interest, but also performs an activity that performs a unique function It is suitable for inducing expression in form.

The method for producing a recombinant protein using the gene of RpoS of the present invention as a fusion partner can overcome the limitations on water solubility and folding of existing fusion partners, and can be widely used for protein medicine and industrial production. .

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Escherichia coli under environmental stress Protein bodies  Change analysis

<1-1> cultured under proteolytic stress In E. coli  Recovery of expressed water soluble protein

In order to give E. coli a stress that inhibits the correct folding of the protein, water-soluble proteins expressed in E. coli cultured in a medium containing GdnHCl showing a proteolytic effect were recovered.

Escherichia coli BL21 cultured in LB medium consisting of 37 g, 130 rpm and 10 g of tryptone, 1 g of yeast extract, and 5 g of NaCl coli K-12) was further cultured in fresh LB medium (control) and LB medium containing 100 mM GdnHCl (experimental group) when OD ₆₀₀ reached 0.5, and then the cell culture was centrifuged at 6000 rpm 4 ° C. The cell precipitate was recovered. After washing the cell precipitate twice with 40 mM Tris-HCl buffer (pH 8.0), 500 μl of crushing solution [lysis buffer; 8M Urea, 4% (w / v) CHAPS, 40 mM Tris, Protease inhibitor cocktail (Roche Diagnostics GmbH, Germany)] and suspended using a Branson sonifier (Branson Ultrasonics Corporation, USA). By crushing. The lysate was centrifuged at 12,000 rpm for 60 minutes at 4 ° C. to separate supernatant and sediment to remove protein aggregates and to separate supernatant. Protein concentration of the isolated supernatant was measured using a Bio-Rad protein assay kit, and 30 mg of the water-soluble protein in the supernatant was rehydrated with a 2 M thiourea. , 8 M urea, 4% (w / v) chaps, 1% (w / v) DTT, 1% (w / v) mobile ampholyte, pH 4.7] 2 Samples for dimensional gel electrophoresis (2-dimmensional polyacrylamide gel electrophoresis; 2D-PAGE) were cryopreserved at -80 ° C.

<1-2> Protein bodies  Two-Dimensional Gel Electrophoresis for Analysis

The protein bodies of the control group and the experimental group obtained by the method of Example <2-1> were analyzed using two-dimensional gel electrophoresis.

The one-dimensional isoelectric point separation process for separating the cryopreserved protein by pI is performed on the cryopreserved protein in a linear IPG (immobilized pH gradient) gel strip (pH 4-7, 17 cm, ReadyStrip, BIO-RAD, USA). After 45 μg aliquot, 500 V, 2 h in Bio-Rad Protein IEF cell electrophoresis system (USA); 1000 V, 30 minutes; 2000 V, 30 minutes; 4000 V, 30 minutes; 8000 V, 70000 VHr (Volt-hours). The IPG gel strip containing proteins separated by pi was equilibrated with 1% DTT and bromophenol blue [epuilibration solution; 50 mM Tris, pH 8.6, 6 M urea, 30% (v / v) glycerol, 2% SDS] for 15 minutes, followed by 2.5% iodoacetamide. Reaction was carried out in an equilibration solution containing 15 minutes. The gel strips equilibrated to separate proteins by molecular weight were subjected to secondary electrophoresis using a PROTEAN II Xi cell system (Bio-Rad, USA) using 12.5% polyacrylamide gel. Electrophoresis was run at 4 ° C. until bromophenol blue reagent reached the end of the gel (30 mA / gel, 12 h).

Rabilloud T, Methods Mol The gel was silver stained according to Biol , 112: 297-305, 1999) and then scanned with a UMAX PowerLook 1100 scanner (UMAX, USA) and image master software version 4.01 (Amersham Biosciences, USA). The change in density per area of each protein spot shown in the gel was measured and analyzed. Based on the results of the analysis, the expression levels of proteins isolated from the control group and the experimental group were compared, and the spot of the protein with a 6-fold increase in the spot volume was selected from the experimental group (FIG. 1).

< Example  2> MALDI - TOF - MS Identification of Escherichia Coli Protein with Increased Water Soluble Expression Under Environmental Stress Using Analysis

The protein spots selected in Example 1 were extracted and deposited in the Korea Basic Science Institute, and identified using MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time Of Flight, MALD-TOF) -MS analysis.

Specifically, the protein spots selected by Example 1 for MALDI-TOF-MS analysis were extracted from silver-stained gels (Gharahdaghi F,et al .,Electrophoresis, 20: 601-605, 1999). Peptide digestion was performed by placing the extracted protein spot in a 25 nM ammonium bicarbonate (pH 8.0) solution containing trypsin (10.15 mg / ml) overnight at 37 ° C. The digested peptide was extracted using 5% (v / v) TFA, 50% (v / v) ACN solution, and the solution containing the peptide extracted using a vacuum centrifuge was repeated three times. Dried. The dried peptide was dissolved in a 50% ACN / 0.1% TFA solution, and deposited in the Korea Basic Science Institute to determine the molecular weight using a MALDI-TOF-MS system (Voyager DE-STR instrument; Biosystems, USA). Peptide mass fingerprints were measured using MS-FIT (http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm) on the Prospector website. Swiss-Prot was used as the MS-FIT database. As a result of protein identification, the protein with 6-fold increase in expression level under 100 mM GdnHCl was identified as E. coli RpoS (Table 1).

Protein Information Gene name Genetic access number ^a Protein name Isoelectric point (pI) / molecular weight (kDa) Relative expression increase ^d % Sequence Similarity  Rpos  P13445 RNA polymerase sigma factor Theoretical value ^b Experimental value ^c  6  18.5%  4.9 / 40.0  5.2 / 39.0

a. Gene access number: An identification number for genetic information retrieval from ExPASy Proteomics Server (http://www.expasy.org/).

b The theoretical values were collected using the Compute pI / Mw tool (http://www.expasy.org/tools/pi_tool.html).

c Experimental values were calculated from two-dimensional electrophoretic gel images.

d The expression level of the control group RpoS was set to 1 to indicate the relative expression level of the experimental group RpoS.

< Example  3> Preparation of Expression Vector Comprising RpoS at the Amino Terminal as a Fusion Partner

An expression vector comprising E. coli protein RpoS (RNA polymerase sigma factor) having increased water soluble expression under environmental stress selected by the method of Examples 1 to 2 was prepared as a fusion partner.

To obtain nucleotides encoding the RpoS gene, primer pairs for PCR amplification of the RpoS gene excluding the stop codon were prepared using sequence information (SEQ ID NO: 45) from 2864581bp to 2865573bp in gi: 49175990 of the Entrez Nucleotide database. . In addition, the sense primer of the primer pair (SEQ ID NO: 1: cat atg agt cag aat acg ctg aaa) has Nde I restriction enzyme recognition sequence, the antisense primer (SEQ ID NO: 2: ctc gag ctc gcg gaa cag cgc ttc), the Xho I It was made to include restriction enzyme recognition sequence. PCR was performed using DNA polymerase buffer solution (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100 Chromosome isolated from Escherichia coli) was added using 100 ng of template DNA and 50 pmol of primer pairs described in SEQ ID NOs: 1 to 2, respectively, followed by Taq DNA polymerase. The reaction conditions were performed 30 times at 95 ° C./30 sec (denatured), 52 ° C./30 sec (annealed), and 72 ° C./60 sec (extension), resulting in Nde I restriction at the 5 ′ end of the amplified DNA fragment. PCR products containing enzyme sites and Xho I restriction enzyme sites at the 3 'end were obtained. The amplified PCR product by restriction enzymes Nde I and Xho I in each treatment was inserted into the restriction enzymes Nde I and Xho I seat of pT7-7 (Novagen, USA) was named this pT7-RpoS.

< Example  4> Expression vector to prepare foreign protein in the form of fusion protein

In order to confirm the increase in water-soluble expression of various foreign proteins using RpoS as a fusion partner, a fusion expression vector including a single expression vector of a foreign protein, a fusion expression vector with a fusion partner, and a protein cleavage enzyme recognition site was prepared.

<4-1> foreign protein alone expression vector

Human minipro-insulin (mp-INS; EF518215: 1-180bp; SEQ ID NO: 46), human epidermal growth factor (EGF; NCBI Nucleotide accession number M15672: 1-165 bp) , Human prepro-ghrelin (ppGRN; NCBI Nucleotide accession number NM; 016362: 109-393), human interleukin-2 (hIL-2; NCBI Nucleotide accession number NM; 000586 116-517), human activation induced cytidine deaminase (AID); NCBI Nucleotide accession number NM; 020661: 77-673bp), human glutamate decarboxylase (GAD) _448-585; SEQ ID NO: 24), also the Pseudomonas footage (Pseudomonas putida ) cutinase (CUT; SEQ ID NO: 47), human ferritin light chain (hFTN-L; NM; 000146: 200-727 bp; SEQ ID NO: 48), human granulocyte colony-stimulation Human granulocyte colony-stimulating factor (G-CSF; NCBI Nucleotide accession number NM; 172219: 131-655 bp) and cold autoinflammatory syndrome Nacht domain, hereinafter Nacht; DNA polymerization comprising 50 pmol each of primer pairs of Table 2 prepared to include Nde I restriction enzyme recognition sequence at 5'-end and 3'-end of Hind III restriction enzyme at 3'-end of foreign protein such as SEQ ID NO. Template DNA in enzyme reaction buffer (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100) 100 ng was added, and then 30 times of PCR were performed using a Taq DNA polymerase at 95 ° C./30 sec (denatured), 52 ° C./30 sec (annealed), and 72 ° C./60 sec (extension).

Specifically, mp-INS, EGF, ppGRN, hIL-2, AID, CUT, G-CSF and hFTN-L amplified the amino acid sequence including the stop codon except for the start codon, and _{448 for} GAD _448-585 . 138 amino acid sequences corresponding to the 585th amino acid (SEQ ID NO: 23), Nacht amplified the 316 amino acid sequence corresponding to the nacht domain of cryopyrin (SEQ ID NO: 24). At this time, in the case of GAD _{448 -585} was prepared a primer to include the Cla I restriction enzyme recognition sequence on the 3'-end.

In addition, mp-INS, EGF, ppGRN , hIL-2, AID, GAD 448 -585, hFTN-L, G-CSF , and template DNA of Nacht is RNeasy mini kit (QIAGEN, USA from a human tissue that the proteins are expressed much ), Extract total RNA, and fill up to 50 µl of distilled water in 1 µg of total RNA and 1 µl of oligo-d (T) (Invitrogen, USA, 0.5 µg / µl), followed by AccuPower RT-premix (Bioneer, Korea). RT-PCR (reverse transcription polymerasechain reaction) reaction. 5 minutes at 70 ° C, 5 minutes at 4 ° C, 60 minutes at 42 ° C, 5 minutes at 94 ° C, and 5 minutes at 4 ° C to obtain cDNA. The mp-INS is human pancreatic tissue, the epithelial cells EGF, ppGRN is human placenta, hIL-2, hFTN-L , G-CSF is human leukocytes and Nacht, AID is a human cell temporal and GAD _{448 -585} of the human hippocampal tissue Cloned from For CUT, Genomic DNA Purification. DNA extracted from Pseudomonas putida was used as template DNA.

Primer sequence Gene name primer SEQ ID NO: Sequence mp-INS sense SEQ ID NO: 3 cat atg ttt gtc aac caa cat Antisense SEQ ID NO: 4 aag ctt tta gtt aca gta gtt c EGF sense SEQ ID NO: 5 cat atg aac tct gac tcc gaa tgc Antisense SEQ ID NO: 6 aag ctt tta acg cag ttc cca cca ppGRN sense SEQ ID NO: 7 cat atg ggc tcc agc ttc ctg Antisense SEQ ID NO: 8 aag ctt tca ctt gtc ggc t hIL-2 sense SEQ ID NO: 9 cat atg gca cct act tca agt Antisense SEQ ID NO: 10 aag ctt tta tca agt cag tgt AID sense SEQ ID NO: 11 cat atg gac agc ctc ttg atg aac Antisense SEQ ID NO: 12 aag ctt tca taa caa aag tcc ca GAD _{448 -585} sense SEQ ID NO: 13 cat atg cgc cac gtt gat gt Antisense SEQ ID NO: 14 atc gat tta taa atc ttg tcc CUT sense SEQ ID NO: 15 cat atg gct ccc ctg ccg gat ac Antisense SEQ ID NO: 16 aag ctt tta aag ccc gcg gcg ct hFTN-L sense SEQ ID NO: 17 cat atg agc tcc cag att cgt Antisense SEQ ID NO: 18 aag ctt tta gtc gtg ctt gag agt G-CSF sense SEQ ID NO: 19 cat atg act cca ctc gga cct g Antisense SEQ ID NO: 20 aag ctt tca tgg ctg tgc aag Nacht sense SEQ ID NO: 21 cat atg act gtg gtg ttc cag Antisense SEQ ID NO: 22 aag ctt tca cag cag gta gta c

The PCR amplification product Nde I / Hind III (the case of GAD _{448 -585} is Nde I / Cla I) was treated with restriction enzymes, Nde I / Hind III of E. coli expression vector pT7 (Novagen, USA) (GAD 448 - _In the case of ₅₈₅ , a single expression vector of a foreign protein was obtained by insertion into the Nde I / Cla I) site.

<4-2> Fusion Expression Vector of a Foreign Protein with a Fusion Partner

mp-INS, EGF, ppGRN, hIL-2, AID, GAD 448 -585, CUT, hFTN-L, Xho I on the 5'-end of the heterologous protein such as G-CSF and Nacht restriction enzyme recognition sequence and a 3 ' DNA polymerase reaction buffer containing 50 pmol of each primer pair of Table 3 prepared to include Hind III restriction enzyme recognition sequence at the end (0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 100 ng of template DNA was added to 20 mM Tris-HCl (pH8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100) and then 95 ° C / 30 seconds (denatured) using Taq DNA polymerase, 52 ° C / A total of 30 PCRs were performed at 30 seconds (annealing) and 72 ° C./60 seconds (extension). Specifically, hFTN-L, mp-INS, EGF, ppGRN, hIL-2, G-CSF, AID, and CUT amplified the amino acid sequence including the stop codon except for the start codon, and _{448 for} GAD _448-585 . 138 amino acid sequences corresponding to the 585th amino acid (SEQ ID NO: 23), Nacht amplified the 316 amino acid sequence corresponding to the nacht domain of cryopyrin (SEQ ID NO: 24). At this time, in the case of GAD _{448 -585} was prepared a primer to include the Cla I restriction enzyme recognition sequence on the 3'-end.

Primer sequence Gene name primer SEQ ID NO: Sequence mp-INS sense SEQ ID NO: 25 ctc gag ttt gtc aac caa cat Antisense SEQ ID NO: 26 aag ctt tta gtt aca gta gtt c EGF sense SEQ ID NO: 27 ctc gag aac tct gac tcc gaa tgc Antisense SEQ ID NO: 28 aag ctt tta acg cag ttc cca cca ppGRN sense SEQ ID NO: 29 ctc gag ggc tcc agc ttc ctg Antisense SEQ ID NO: 30 aag ctt tca ctt gtc ggc t hIL-2 sense SEQ ID NO: 31 ctc gag gca cct act tca agt Antisense SEQ ID NO: 32 aag ctt tta tca agt cag tgt AID sense SEQ ID NO: 33 ctc gag gac agc ctc ttg atg aac Antisense SEQ ID NO: 34 aag ctt tca taa caa aag tcc ca GAD _{448 -585} sense SEQ ID NO: 35 ctc gag cgc cac gtt gat gt Antisense SEQ ID NO: 36 atc gat tta taa atc ttg tcc CUT sense SEQ ID NO: 37 ctc gag gct ccc ctg ccg gat ac Antisense SEQ ID NO: 38 aag ctt tta aag ccc gcg gcg ct hFTN-L sense SEQ ID NO: 39 ctc gag agc tcc cag att cgt Antisense SEQ ID NO: 40 aag ctt tta gtc gtg ctt gag agt G-CSF sense SEQ ID NO: 41 ctc gag act cca ctc gga cct g Antisense SEQ ID NO: 42 aag ctt tca tgg ctg tgc aag Nacht sense SEQ ID NO: 43 ctc gag act gtg gtg ttc cag Antisense SEQ ID NO: 44 aag ctt tca cag cag gta gta c

The PCR amplification product Xho I / Hind III (the case of GAD _{448 -585} are Xho I / Cla I) was treated with restriction enzymes, the expression vector pT7-RpoS Xho of production by the method of Example 3 I / Hind III to give a fusion expression vector for the fusion partner of the foreign protein by inserting a place (in the case of GAD _448-585 is Nde I / Cla I). Plasmids produced by the above method were pT7-RpoS :: FTN-L, pT7-RpoS :: IL-2, pT7-RpoS :: EGF, pT7-RpoS :: G-CSF, pT7-RpoS :: AID, pT7 -RpoS :: CUT, pT7-RpoS :: ppGRN, pT7-RpoS :: GAD448-585 and pT7-RpoS :: Nacht.

<4-3> Expression vector for purification comprising 6 histidines

Sense to include the Xho I restriction enzyme recognition sequence at the 5'-end of the foreign protein CUT containing the stop codon and the polynucleotide and Hind III restriction enzyme recognition sequences encoding 6 histidines at the 3'-end, except for the start codon. Buffer solution for DNA polymerase reaction containing 50 pmol of primer (SEQ ID NO: 49: ctc gag gct ccc ctg ccg gat ac) and antisense primer (SEQ ID NO: 50: aag ctt tta gtg atg gtg atg gtg atg aag ccc gcg ct) 100 ng of template DNA was added to 0.25 mM dNTPs; 50 mM KCl; 10 mM (NH ₄ ) ₂ SO ₄ ; 20 mM Tris-HCl (pH 8.8); 2 mM MgSO ₄ ; 0.1% Triton X-100) PCR was performed a total of 30 times at 95 ° C./30 sec (denatured), 52 ° C./30 sec (annealed), and 72 ° C./60 sec (extension) using DNA polymerase. Specifically, the amino acid sequence including the stop codon was amplified except for the start codon.

PCR products of the CUT were treated with restriction enzymes Xho I and Hind III, respectively, and inserted into the Xho I and Hind III sites of the expression vector containing RpoS as a fusion partner at the amino terminus of Example 3, which were then expressed as pT7-RpoS :: CUT. It was named -His6.

< Example  5> Water soluble expression of recombinant protein

E. coli was transformed and cultured with a single expression vector of a foreign protein prepared by the method of Example 4, a fusion expression vector with a fusion partner, and a purified expression vector containing 6 histidines, and then expression of the recombinant protein with IPTG. By induction, the effect of water-soluble expression by the fusion partner of the present invention was confirmed.

<5-1> Escherichia Coli Transformation and Recombinant Protein Expression

Method described by Hanahan (Hanahan D, DNA Cloning vol. 1 109-135, IRS press 1985) were transformed into E. coli vectors of Example 4.

Specifically, the vectors of Example 4 were transformed into E. coli BL21 (DE3) treated with CaCl ₂ by a heat shock method, and then cultured in a medium containing ampicillin to transform the expression vector into ampicillin resistance. Representative colonies were selected. E. coli transformed with the fusion expression vector pP7-RpoS with the fusion partner was named BL21 (DE3): pP7-RpoS. A portion of the spawn cultures in which the colonies were cultured in LB medium overnight was inoculated in LB medium containing 100 mg / ml ampicillin and then incubated at 37 ° C. at 200 rpm. When OD ₆₀₀ of the culture reached 0.5, IPTG was added (1 mM) to induce the expression of recombinant genes. After addition of IPTG, the cells were further incubated for 3 to 4 hours under the same conditions.

<5-2> SDS - PAGE Identification of Recombinant Protein Produced by Solubility

E. coli cultured by the method of Example 5-1 was centrifuged at 6,000 rpm for 5 minutes to recover the cell precipitate, and then suspended in 5 ml of crushed solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA) Crushing was performed using a crusher (Branson sonifier, Branson Ultrasonics Corporation, USA). After crushing, the mixture was centrifuged at 13,000 rpm for 10 minutes, and the supernatant and the insoluble aggregate were separated. Protein concentration of the isolated supernatant was measured using a Bio-Rad protein assay kit (USA). In addition, the supernatant and insoluble aggregates were mixed with 5 × SDS (0.156M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT), respectively, 1: 4 and boiled at 100 ° C. for 10 minutes. The boiled sample was loaded into a well of 10% SDS-PAGE gel, the sample was developed at 125 V for 2 hours, the gel was stained by Coomassie staining, and then decolorized. -Rad, USA) and the solubility (%) was calculated according to Equation (1).

As a result, it was confirmed that the expression of foreign proteins fused with RpoS was more in water-soluble expression than insoluble aggregates (FIG. 3). In addition, it was confirmed that the solubility in the foreign protein fused with RpoS is significantly increased than the water-soluble expression of the foreign protein expressed alone (FIG. 4).

<5-3> Fusion with RpoS Cutinase  Enzyme activity

The hydrolytic activity of the RpoS fusion-expressed cutinase (hereinafter RpoS :: CUT) obtained through Examples 5-1 to 5-2 was measured.

106.7 μl of 0.1M phosphate buffer (pH 8.0) and 13.3 μl of Triton X-100 (4 g / L) were added to a 96-well microplate, followed by E. coli BL21 (DE3) as an unpurified RpoS :: CUT supernatant and a control. 13.3 μl of supernatant) were added, respectively. The hydrolysis reaction was started by adding 66.7 μl of substrate (p-nitrophenyl palmitate) and PNB (p-nitrophenyl butyrate, 6.6 mM; Sigma, USA) to the solution and reacted at 37 ° C. for 20 minutes. After the reaction was started, the change in absorbance at 415 nm wavelength was measured every minute (Bio-Rad microplate reader, USA).

As a result, it was confirmed that the water-soluble recombinant protein RpoS :: CUT has PNP-specific degradation capacity (FIG. 5).

<5-4> Purification of Recombinant Protein Using Histidine Tag

The method described by Hanahan of Example 5-1 (Hanahan D, DNA ) for a purified expression vector containing six histidines prepared by the method of Example 4-3 Cloning vol. 1 109-135, IRS press 1985) was transformed into E. coli BL21 (DE3), and the expression of recombinant genes was induced. The transformed BL21 (DE3) was disrupted by the method of Example 5-2, and then the supernatant and the insoluble aggregate were separated by centrifugation.

From the supernatant, recombinant protein RpoS :: CUT-His ₆ was purified by affinity chromatography. Specifically ProBond resin that is filled with the metal ions (Ni ^{+ 2)} column (Invitrogen, USA) with binding buffer [binding buffer; pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 10 mM imidazole], and the washed column contains RpoS :: CUT-His ₆ . Supernatants were bound at 4 ° C. Thereafter, 8 ml of washing buffer; pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 50 mM imidazole] and washed twice with elution buffer; RpoS :: CUT-His ₆ was recovered using pH 8.0, sodium phosphate, 300 mM sodium chloride (NaCl), 250 mM imidazole].

Amicon Ultra-4 centrifugal filter (Millipore, USA) was used to remove imidazole present in the elution buffer containing RpoS :: CUT-His ₆ . PBS buffer [PBS buffer; 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM sodium monophosphate (Na ₂ HPO ₄ ), 2 mM potassium monophosphate (KH ₂ PO ₄ ), pH 7.4] Solution exchange was performed.

Separate supernatant and purified RpoS :: CUT-His ₆ with 5 × SDS (0.156M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% Glycerol, 37.5 mM DTT), respectively, 1: 4 Boiled for a minute. Boiled samples were loaded into wells of a 10% SDS-PAGE gel, the samples were developed at 125 V for 2 hours, and the gels were stained by Coomassie staining and then decolorized to confirm the expression level of each recombinant protein.

In addition, purified RpoS :: CUT-His ₆ was analyzed using high-performance liquid chromatography (HPLC). Specifically, an LC-20A Prominence model (Shimadzu Co. Ltd., Japan) and a Shim-pack CLC-NH ₂ column (60 × 150 mm, Shimadzu Co. Ltd., Japan) were used as analytical instruments. 10% acetonitrile (SIGMA, USA) containing 0.1% of trifluoroacetic acids (SIGMA, USA) was equilibrated by flowing 1 mL / min. Thus, RpoS :: CUT-His ₆ purified by the above method was injected into the equilibrated column, and eluted from the column by flowing 5% acetonitrile at a rate of 1 ml / min for 15 minutes. Elution profiles were detected using a 215 nm wavelength.

As a result, it was confirmed that only RpoS :: CUT-His ₆ was purified from the aqueous supernatant (FIG. 6-1). HPLC analysis confirmed that there was one peak (Fig. 6-2).

1 is a result of analyzing the change of E. coli protein body under environmental stress:

A: Results of 2-D PAGE (2-dimensional polyacrylamide gel electrophoresis)

B: Increase of RpoS expression by GdnHCl

2 is a cleavage map of a fusion expression vector comprising a foreign protein alone expression vector, a fusion expression vector with a fusion partner, and a protein cleavage enzyme recognition site:

A: cleavage map of a single expression vector

B: Cleavage map of fusion expression vector

Fig. C: cleavage map of a purified expression vector comprising six histidines

Figure 3 is a result of comparing the expression in the water-soluble supernatant (S) and insoluble aggregate (IS) of the recombinant protein fusion-expressed RpoS.

Figure 4 is a graph comparing the water-soluble expression of the recombinant protein expressed alone and recombinant RpoS fusion expression.

Figure 5 shows the PNB (●) and PNP (○) of the unrefined water-soluble protein of E. coli, which does not express the cutinase, and the unrefined water-soluble protein of E. coli, which expresses the recombinant protein expressed by RpoS fusion in the form of RpoS :: CUT. The result of measuring the resolution of is shown.

6 is a diagram showing the result of purification of recombinant protein using His tag:

Figure A: Purification results using the SDS-PAGE method

M: protein marker

1: Unrefined aqueous supernatant containing RpoS :: CUT-His6

2 and 3: Purified RpoS :: CUT-His6

B: Purification results using HPLC

<110> KOREA UNIVERSITY Industry & Academy Collaboration Foundation <120> PREPARATION METHOD OF RECOMBINANT PROTEIN BY USE OF RpoS AS A          FUSION EXPRESSION PARTNER <130> 7P-08-24 <160> 50 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RpoS Sense Primer <400> 1 catatgagtc agaatacgct gaaa 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RpoS Antisense Primer <400> 2 ctcgagctcg cggaacagcg cttc 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mp-INS Sense Primer <400> 3 catatgtttg tcaaccaaca t 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mp-INS Antisense Primer <400> 4 aagcttttag ttacagtagt tc 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF Sense Primer <400> 5 catatgaact ctgactccga atgc 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF Antisense Primer <400> 6 aagcttttaa cgcagttccc acca 24 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ppGRN Sense Primer <400> 7 catatgggct ccagcttcct g 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ppGRN Antisense Primer <400> 8 aagctttcac ttgtcggct 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2 Sense Primer <400> 9 catatggcac ctacttcaag t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2 Antisense Primer <400> 10 aagcttttat caagtcagtg t 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AID Sense Primer <400> 11 catatggaca gcctcttgat gaac 24 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AID Antisense Primer <400> 12 aagctttcat aacaaaagtc cca 23 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAD448-585 Sense Primer <400> 13 catatgcgcc acgttgatgt 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAD448-585 Antisense Primer <400> 14 atcgatttat aaatcttgtc c 21 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CUT Sense Primer <400> 15 catatggctc ccctgccgga tac 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CUT Antisense Primer <400> 16 aagcttttaa agcccgcggc gct 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L Sense Primer <400> 17 catatgagct cccagattcg t 21 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L Antisense Primer <400> 18 aagcttttag tcgtgcttga gagt 24 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G-CSF Sense Primer <400> 19 catatgactc cactcggacc tg 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> G-CSF Antisense Primer <400> 20 aagctttcat ggctgtgcaa g 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nacht Sense Primer <400> 21 catatgactg tggtgttcca g 21 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Nacht Antisense Primer <400> 22 aagctttcac agcaggtagt ac 22 <210> 23 <211> 948 <212> DNA <213> Human GAD448-585 <400> 23 accgtggtgt tccagggggc ggcagggatt gggaaaacaa tcctggcccg gaagatgatg 60 ttggactggg cgtcggggac actctaccaa gacaggtttg actatctgtt ctatatccac 120 tgtcgggagg tgagccttgt gacacagagg agcctggggg acctgatcat gagctgctgc 180 cccgacccaa acccacccat ccacaagatc gtgagaaaac cctccagaat cctcttcctc 240 atggacggct tcgatgagct gcaaggtgcc tttgacgagc acataggacc gctctgcact 300 gactggcata aggccgagcg gggagacatt ctcctgagca gcctcatcag aaagaagctg 360 cttcccgagg cctctctgct catcaccacg agacctgtgg ccctggagaa actgcagcac 420 ttgctggacc atcctcggca tgtggagatc ctgggtttct ccgaggccaa aaggaaagag 480 tacttcttca agtacttctc tgatgaggcc caagccaggg cagccttcag tctgattcag 540 gagaacgagg tcctcttcac catgtgcttc atccccctgg tctgctggat cgtgtgcact 600 ggactgaaac agcagatgga gagtggcaag agccttgccc agacatccaa gaccaccacc 660 gcggtgtacg tcttcttcct ttccagtttg ctgcagcccc ggggagggag ccaggagcac 720 ggcctctgcg cccacctctg ggggctctgc tctttggctg cagatggaat ctggaaccag 780 aaaatcctgt ttgaggagtc cgacctcagg aatcatggac tgcagaaggc ggatgtgtct 840 gctttcctga ggatgaacct gttccaaaag gaagtggact gcgagaagtt ctacagcttc 900 atccacatga ctttccagga gttctttgcc gccatgtact acctgctg 948 <210> 24 <211> 414 <212> DNA <213> nacht domain of cryopyrin <400> 24 cgccacgttg atgtttttaa actatggctg atgtggaggg caaaggggac taccgggttt 60 gaagcgcatg ttgataaatg tttggagttg gcagagtatt tatacaacat cataaaaaac 120 cgagaaggat atgagatggt gtttgatggg aagcctcagc acacaaatgt ctgcttctgg 180 tacattcctc caagcttgcg tactctggaa gacaatgaag agagaatgag tcgcctctcg 240 aaggtggctc cagtgattaa agccagaatg atggagtatg gaaccacaat ggtcagctac 300 caacccttgg gagacaaggt caatttcttc cgcatggtca tctcaaaccc agcggcaact 360 caccaagaca ttgacttcct gattgaagaa atagaacgcc ttggacaaga ttta 414 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mp-INS Sense Primer <400> 25 ctcgagtttg tcaaccaaca t 21 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> mp-INS Antisense Primer <400> 26 aagcttttag ttacagtagt tc 22 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF Sense Primer <400> 27 ctcgagaact ctgactccga atgc 24 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EGF Antisense Primer <400> 28 aagcttttaa cgcagttccc acca 24 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ppGRN Sense Primer <400> 29 ctcgagggct ccagcttcct g 21 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ppGRN Antisense Primer <400> 30 aagctttcac ttgtcggct 19 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2 Sense Primer <400> 31 ctcgaggcac ctacttcaag t 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hIL-2 Antisense Primer <400> 32 aagcttttat caagtcagtg t 21 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AID Sense Primer <400> 33 ctcgaggaca gcctcttgat gaac 24 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AID Antisense Primer <400> 34 aagctttcat aacaaaagtc cca 23 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAD448-585 Sense Primer <400> 35 ctcgagcgcc acgttgatgt 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAD448-585 Antisense Primer <400> 36 atcgatttat aaatcttgtc c 21 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CUT Sense Primer <400> 37 ctcgaggctc ccctgccgga tac 23 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CUT Antisense Primer <400> 38 aagcttttaa agcccgcggc gct 23 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L Sense Primer <400> 39 ctcgagagct cccagattcg t 21 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hFTN-L Antisense Primer <400> 40 aagcttttag tcgtgcttga gagt 24 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> G-CSF Sense Primer <400> 41 ctcgagactc cactcggacc tg 22 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> G-CSF Antisense Primer <400> 42 aagctttcat ggctgtgcaa g 21 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Nacht Sense Primer <400> 43 ctcgagactg tggtgttcca g 21 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Nacht Antisense Primer <400> 44 aagctttcac agcaggtagt ac 22 <210> 45 <211> 993 <212> DNA <213> Escherichia coli RpoS <400> 45 ttactcgcgg aacagcgctt cgatattcag cccctgcgtt tgcaggattt cgcgcaaacg 60 gcgcaggcct tcaacctgaa tctggcgaac acgttcacgg gtgaggccaa tttcacgacc 120 tacatcttcc agtgttgccg cttcgtaccc cagcaaaccg aatcgacgtg ccagcacttc 180 acgctgtttg gcgttcagct cgaacagcca tttgacgatg ctctgcttca tatcgtcatc 240 ttgcgtggta tcttccggac cgttctcttt ttcatcggcc aggatgtcca gcaacgcttt 300 ttcggaatca ccacccagcg gggtgtctac cgaggtaatg cgctcgttaa gacgaagcat 360 acggctgacg tcatcaactg gcttatccag ttgctctgcg atctcttccg cacttggttc 420 atggtccagc ttatgggaca actcacgtgc ggttcgcagg taaacgttca gctcctttac 480 gatgtgaatc ggcaaacgaa tagtacgggt ttggttcata atcgcccgtt caatcgtctg 540 gcgaatccac caggttgcgt atgttgagaa gcggaaacca cgttccgggt caaacttctc 600 taccgcgcgg atcagcccca ggttgccctc ttcgataagg tccagcaacg ccagaccacg 660 attgccataa cggcgggcaa tttttaccac cagacgcaag ttactctcga tcatccggcg 720 gcgagaggcg acatctccac gcagtgcgcg acgcgcaaaa taaacttctt cttcggccgt 780 taacagtggt gaataaccaa tctcaccaag gtaaagctga gtcgcgtcca acacacgctg 840 tgtggctccc tgcgataaca gttcctcttc ggccaaatcg ttatcactgg gttcctgttc 900 tactaaggcc ttttcgtcaa aaacctcaac tccgttctca tcaaattccg catcttcatt 960 taaatcatga actttcagcg tattctgact cat 993 <210> 46 <211> 180 <212> DNA <213> human minipro-insulin <400> 46 tttgtcaacc aacatttatg tggatcacat ttagtagagg ctttgtatct tgtttgtggt 60 gaacgtggat ttttctatac acctaagaca cgtagatctc ctaatggaaa acgtggtatt 120 gttgaacaat gctgtacatc aatctgttca ttgtatcaac ttgagaacta ctgtaactaa 180                                                                          180 <210> 47 <211> 528 <212> DNA <213> human ferritin light chain <400> 47 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aattggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 ctcatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 gggctgggcg agtatctctt cgaaaggctc actctcaagc acgactaa 528 <210> 48 <211> 780 <212> DNA <213> Pseudomonas putida cutinase <400> 48 atggctcccc tgccggatac accgggagcg ccatttccgg ctgtcgccaa tttcgaccgc 60 agtggcccct acaccaccag cagccagagc gaggggccga gctgtcgcat ctatcggccc 120 cgcgacctgg gtcagggggg cgtgcgtcat ccggtgattc tctggggcaa tggcaccggt 180 gccgggccgt ccacctatgc cggcttgcta tcgcactggg caagccacgg tttcgtggtg 240 gcggcggcgg aaacctccaa tgccggtacc gggcgggaaa tgctcgcctg cctggactat 300 ctggtacgtg agaacgacac cccctacggc acctattccg gcaagctcaa taccgggcga 360 gtcggcactt ctgggcattc ccagggtggt ggcggctcga tcatggccgg gcaggatacg 420 agggtgcgta ccacggcgcc gatccagccc tacaccctcg gcctggggca cgacagcgcc 480 tcgcagcggc ggcagcaggg gccgatgttc ctgatgtccg gtggcggtga caccatcgcc 540 tttccctacc tcaacgctca gccggtctac cggcgtgcca atgtgccggt gttctggggc 600 gaacggcgtt acgtcagcca cttcgagccg gtcggtagcg gtggggccta tcgcggcccg 660 agcacggcat ggttccgctt ccagctgatg gatgaccaag acgcccgcgc taccttctac 720 ggcgcgcagt gcagtctgtg caccagcctg ctgtggtcgg tcgagcgccg cgggctttaa 780                                                                          780 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CUT-His6 Sense Primer <400> 49 ctcgaggctc ccctgccgga tac 23 <210> 50 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> CUT-His6 Antisense primer <400> 50 aagcttttag tgatggtgat ggtgatgaag cccgcgct 38  

Claims (9)

1) preparing an expression vector linking a gene of RpoS (RNA polymerase sigma factor) and a gene of a foreign protein as a fusion partner; 2) preparing a transformant by introducing the expression vector into a host cell; And, 3) culturing the transformant to induce the expression of the recombinant protein and the method of producing a recombinant protein consisting of obtaining it. The method of claim 1, wherein the gene of RpoS is set forth in SEQ ID NO: 45. The method of claim 1, wherein the foreign protein has a biological activity selected from the group consisting of antigens, antibodies, cell receptors, enzymes, structural proteins, serum and cellular proteins. An expression vector for recombinant protein production comprising a gene of RpoS and a gene of foreign protein as a fusion partner. The expression vector according to claim 4, which is represented by the cleavage map of FIG. The expression vector according to claim 4, wherein the fusion partner includes an RpoS gene and a foreign protein gene. The expression vector according to claim 4, wherein a polynucleotide encoding a protein cleavage site is linked between the RpoS gene and the foreign protein gene. The transformant transformed with the expression vector of claim 4. A recombinant fusion protein produced by the method of claim 1.

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