KR19990009995A - Novel human growth hormone-releasing factor-human epidermal growth factor fusion gene, expression vector thereof, and method for manufacturing human growth hormone-releasing factor using the same - Google Patents

Abstract

The present invention relates to a new method for producing human growth hormone release factor (hGRF) to promote the secretion of human growth hormone. The present invention is a hEGF-hGRF fusion gene synthesized by fusing the hGRF gene with a human epidermal growth factor (hEGF) gene by a gene recombination method, an expression vector capable of expressing it in large amounts in E. coli and secreting it extracellularly The present invention relates to a method for efficiently producing hGRF protein by culturing pEGRF and microorganisms transformed with the present expression vector, and then separating and obtaining the hEGF-hGRF fusion protein, using the hEGF-hGRF fusion gene and the expression vector of the present invention. Pure hGRF protein can be easily isolated and purified from cell culture media.

Description

Novel human growth hormone-releasing factor-human epidermal growth factor fusion gene, expression vector thereof, and method for manufacturing human growth hormone-releasing factor using the same

1 shows a process for preparing a plasmid vector pGBIle containing a gene in which methionine, amino acid No. 27, in the human growth hormone release factor (hGRF) gene is substituted with isoleucine.

Figure 2 is a human growth hormone by introducing the hGRF gene on the pGBlle vector of Figure 1 in the carboxy terminal region of the human epidermal growth factor in the expression vector pTE105 containing the ompA leader sequence of E. coli and human epidermal growth factor (hEGF) gene It shows the manufacturing process of the expression vector pEGRF in which the release factor (hGRF) protein is expressed as a protein fused with human epidermal growth factor (hEGF).

Figure 3 shows the result of the expression of the expression vector pEGRF transformed into E. coli JM101 recombinant E. coli transformed into a human growth hormone release factor (hGRF) in the human epidermal growth factor (hEGF) fused form results Polyacrylamide gel electrophoresis and western blotting are shown.

[Objective of the invention]

The present invention relates to a novel method for producing a human growth hormone releasing factor (hereinafter abbreviated as hGRF ') that promotes the secretion of human growth hormone.

More specifically, the present invention expresses and secretes the hGRF protein in the form of a fusion with a human epidermal growth factor (hereinafter abbreviated as hEGF) protein to obtain hEGF-hGRF fusion protein in culture. As a method for producing hGRF protein by cleaving the same, a gene recombination method is used to inhibit degradation of protease in E. coli cells until hGRF is expressed in large amounts in E. coli and secreted into the cell. A novel hEGF-hGRF fusion gene prepared by fusing the hGRF gene and the hGRF gene, an expression vector pEGRF capable of expressing it in large quantities in Escherichia coli, and an E. coli transformed with the expression vector, and culturing it to efficiently hGRF It relates to a manufacturing method to produce.

By using the hEGF-hGRF fusion gene and the expression vector of the present invention, pure hGRF protein can be easily isolated and purified from the cell culture, and can be obtained with high yield.

[Technical Field to which the Invention belongs and Conventional Technology in the Field]

Human Growth Hormone-releasing Factor (hGRF) is a polypeptide of about 5040 Daltons consisting of 44 amino acids and is a hormone that is secreted from the hypothalamus of human and acts on the pituitary gland to promote the release of human growth hormone (Gillemin, et al., Science , 218, 585-587, 1982).

This hGRF shows the activity of the hormone without specific species, so in vivo (in vivo) To promote the growth hormone secretion of various farm animals such as cattle, pigs, sheep and chickens. The secretion of growth hormone promotes energy metabolism in breeding animals, leading to weight gain and lactation (cow, ewe), and reducing fat to improve meat quality. Attention is focused on (Pomnier,et al. , J. Anim Sci. , 168, 1291-1298, 1990; Etienne, Metal. ,J. Anim. Sci. , 70, 2212-2220, 1992; Lapierre,et al. , J. Anim. Sci. ,-764-772, 1992.).

There have also been many attempts to use hGRF in the treatment of diseases. Specifically, hGRF may be widely applied to the treatment of cartilage fractures, osteoporosis, wounds, and burns of large areas, including those used for the treatment of dwarfism and delayed development (Borges, JL, et. al ., Lancet , 16, 119-123, 1983; Garrel, DR, et al., J, Surgical Research , 51, 297-302, 1991). In addition, hGRF has recently promoted ovulation to increase pregnancy rates (Tuladi, et al ., Human Reproduction , Vol. 8, No. 4, 525-527, 1993; Volpe, A. et al ., Human Reproduction , Vol. 6 , No. 9, 1128-1232, 1991), it has been shown to increase appetite in patients with loss of appetite (Vaccarino, FJ, et al., Biol. Psychiatry , 35, 446-451, 1994). The prospect of having is growing.

Since Gilille's first discovery of the minoic acid sequence of hGRF from extracts from human pancreatic tumors in 1982, a number of studies have been conducted to artificially synthesize hormones. Since human growth hormone releasing factor (hGRF) is a relatively short single polypeptide, hGRF and its derivatives can be easily prepared by chemical synthesis methods (Korean Patent Publication No. 92-6564), and in fact, some synthetic hGRFs are used for diagnostic reagents. Has been sold. However, the chemical synthesis of the protein is not only low in yield but also expensive, and there are many problems in mass production of hGRF.

Recently, many attempts have been made to produce hGRF by genetic recombination. In 1985, Calvador et al. Attempted intracellular expression of hGRF in Escherichia coli using the P _L promoter of bacteriophage lambda (Cravador, et al., Biochimie, 67, 829-834, 1985).

However, hGRF protein has a problem of being easily degraded by proteases in E. coli. In order to solve this problem, studies have been conducted to prevent the degradation of hGRF by the protease by fusing a gene encoding another protein stable to the protease with the hGRF gene.

In 1988, Villa et al. Incorporated the tryptophan E gene into the GRF gene to express GRF intracellularly in the form of a fusion protein (S. Villa., Et al., Eur. J. Biochem ., 171, 137-141, 1988). ). Also, in 1987, Anba et al. Deficient the phosphate binding protein S gene beside the GRF gene and expressed it intracellularly in the form of a fusion protein (Anba, et al., 53, 219-226,1987), and in 1992 Kura et al. Also lacked the dihydroreductase (DHFR) gene beside the hGRF gene and expressed it intracellularly in the form of a fusion protein (lwakara, et al., 111, 37-45, 1992).

However, the above methods are a method of expressing hGRF protein in a cell, and when a foreign protein is present in E. coli, it is rapidly decomposed by a protease. In addition, to recover the hGRF protein, E. coli must be crushed to collect the protein, which leads to complicated purification procedures, reduced yield, much time and effort for separation and purification, and E. coli in the process of recovering the protein by crushing E. coli. Since endotoxin may be incorporated as an impurity, there is a problem that an additional process for completely removing endotoxin is required.

In order to solve the above-mentioned problems, the present inventors expressed a large amount of 질 1 protein in E. coli in the form of a coagulated protein, and then released it out of E. coli cells outside the rye and outside the cells. A long study has been conducted to suppress the degradation and to prevent the incorporation of intracellular protein impurities by facilitating dilution of hGRF protein from cells without diluting hGRF protein.

As a result, the gene of hEGF, which is excellent in expression and highly stable to the protease of Escherichia coli, binds to hGRF gene so that hGRF protein is expressed in E. coli and maintained until it is secreted extracellularly. Synthesis of the fusion gene, expression vector 릍 gene recombination method that can express the fusion protein extracellularly, by culturing the microorganism transformed with the expression vector can be produced in large quantities in the culture medium hEGF-hGRF fusion protein The present invention has been developed to complete the present invention. When the fusion protein prepared according to the present invention is treated with a bromide (cyanobromide, hereinafter abbreviated as CNBr), the hEGF protein hGRF protein difference is cleaved to facilitate recovery of pure hGRF protein.

[Technical Challenges to Invent]

The present invention is a method for producing hGRF protein by expressing and secreting hGRF protein in the form of a fusion protein to the periplasmic region of the cell membrane and extracellularly,

The aim is to provide a novel hEGF-hGRF fusion gene designed to encode a stable protein that is expressed in large amounts in E. coli and is not degraded to E. coli's protease until the expressed protein is secreted extracellularly. The hEGF-hGRF fusion gene of the present invention inhibits the hGRF protein from being degraded by the protease until it is secreted into the cell after mass expression of the hEGF-hGRF fusion protein in soluble form. To get it.

The present invention relates to a plasmid pGB002 containing the hGRF gene [KPAC Accession No .: KFCC-1094, Korean Patent 1996 Patent Application No. 53539, a new human growth hormone releasing factor (hGRF) gene, its expression vector and hGRF using the same See Production Method.] A plasmid vector pGBlle prepared by replacing a sequence encoding amino acid methionine, amino acid 27 of the hGRF gene, with isoleucine is provided. In the hGRF gene of the present invention, the hGRF protein is not cleaved by CNBr when the hEGF-hGRF fusion protein is cleaved with CNBr because methionine is substituted with isoleucine.

It is also an object of the present invention to provide an expression vector containing a hEGF-hGRF fusion gene to express a large amount of hEGF-hGRF fusion protein in a soluble form to secrete it into cell culture.

Specifically, the present invention provides a vector of expression of pTE105 which expresses a large amount of hEGF under ompA leader sequence and secretes it extracellularly (see Korean Patent Application No. 93-6979, Human Epidermal Growth Factor (hEGF) Expression Vector and Preparation of hEGF Using the Same). The hGRF (Met271le) gene in the pGB1le plasmid vector is inserted into the carboxy terminal region of the hEGF gene to provide an expression vector pEGRF capable of expressing the hEGF-hGRF fusion protein by regulation of the tac promoter.

In addition, the present invention is ompA leader sequence-universal translation stop sequence- trpA transcription stop sequence, the expression using the expression cassette inserted hEGF gene is regulated transcription by the tac promoter between the ompA leader sequence and universal translation stop sequence Its purpose is to provide a method for producing a vector. Specifically, a nucleic acid fragment of 2917 base of the pTE105 expression vector obtained by cleaving the carboxy terminal region of the hEGF gene with Afl ll and Pac l restriction enzyme and a fragment of 138 bases of Mbo ll and Pac l from the pGBlle plasmid vector were obtained. Synthesized nucleic acid linkers of the above to prepare the expression vector pEGRF of the present invention.

The transformant obtained by transforming E. coli strain JM1O1 with the expression vector pEGRF of the present invention was deposited on March 27, 1997 to the Korean spawn association (accession number: KFCC-10963).

It is also an object of the present invention to provide a method for culturing the E. coli transformants to obtain E. coli cells and to produce irobuti hGRF protein.

Through the above process, the pure human growth hormone release factor can be easily separated and purified.

[Configuration and Function of Invention]

The present invention is a hEGF- fusion of a new human epidermal growth factor (hEGF) gene and a human growth hormone releasing factor (hGRF) gene designed to be easily expressed in large quantities in E. coli and stable until the expressed protein is secreted extracellularly. hGRF fusion gene is provided.

In the present invention, the fusion partner was selected in consideration of the following three points.

First, the protein can be expressed in large quantities. Second, the expressed protein has a stable structure in the cell. Third, the expressed protein is secreted extracellularly in a soluble form, thereby facilitating recovery and purification of the fusion protein. You should be able to.

Using the hEGF gene as a fusion partner in the present invention as a fusion partner that satisfies these conditions has the following five advantages.

First, since there is a large amount of hEGF expression in Escherichia coli, hEGF-hGRF fusion protein can be expressed in large quantities. Second, hEGF is a small protein consisting of 53 amino acids and has a fairly stable structure for host protease. In addition, the expressed hEGF-hGRF fusion protein is stably preserved intracellularly during the secretion of extracellularly. Third, the recovery and purification of the fusion protein is carried out by expressing it in a soluble form by adding an ompA leader sequence to the hEGF gene. Fourth, since the titration method (ELISA, RIA) of hEGF is established, analysis and quantification are possible in the purification process of fusion protein. See Korean Patent Application Nos. 93-6978, 93-6979, 93-6980 filed by the inventors).

Specifically, by fusing the hGRF gene using the asparagine-methionine linker gene cleavable by CNBr to the carboxy terminus of the hEGF gene (Korean Patent Application No. 93-6979), which is expressed in excess of 400 mg / liter on average, While preventing hGRF degradation, the hERF protein and hGRF protein are cleaved from the fusion protein when treated with CNBr to facilitate separation and purification.

The present invention provides a plasmid vector pGBlle that contains a hGRF gene in which methionine is substituted with isoleucine.

The 27th amino acid of the natural hGRF protein is methionine, which is cleaved during CNBr treatment during protein purification, so that the 27th amino acid, methionine, is replaced with isoleucine to prevent the hGRF protein from being damaged by CNBr treatment. This was done.

First, to obtain the hGRF gene, the plasmid vector pGB002 (Korean Patent Application No. 96-53539) containing the hGRF gene was used.

Specifically, the plasmid vector pGB002 was cleaved with restriction enzymes Bsm AI and Kpn I to obtain a 112 bp hGRF gene segment, and the same plasmid vector was cleaved with Kpn I and Pst I restriction enzymes to obtain a gene segment having a size of 2952 bp. A 14 bp synthetic linker having the base sequences of SEQ ID NOS: 1 and 2 was prepared to replace matchonin, the 27th amino acid in the hGRF protein, with isoleucine.

The hGRF gene fragment, the plasmid vector pGB002, which was cleaved with Kpn I and Pst I restriction enzymes, and a 2952 bp gene fragment, was synthesized by T4 DNA ligase by reacting a 14 bp synthetic linker gene designed to replace matchonine with isoleucine in the hGRF protein. After conjugation, it was transformed into E. coli XL1-blue by one method (Hanahan, et al ., DNA CIoning, Vol 1, A Practica1 Approach, IRL Press, 1985, 109-135P) and the plasmid vector pGBlle. Get

The present invention inserts the hGRF (Met271le) gene in the plasmid Vecti pGBlle into the carboxy terminal region of the hEGF gene in pTE105, which is an expression vector in which a large amount of hEGF was expressed in Escherichia coli, and is expressed in a fused form of hEGF and hGRF. An expression vector pEGRF prepared to be provided.

Specifically, in order to fuse the hGRF gene to the hEGF gene, the carboxy terminal portion of the hEGF gene present in the hEGF expression vector pTE105 was cut with the restriction enzyme Afl II and cut with the restriction enzyme Pac I to cut a 2917 bp gene segment enriched with the hEGF gene. Get In this process, six amino acids at the carboxy terminus of hEGF are removed, resulting in a structure having 47 amino acids. However, the cleavage of the six amino acids at the carboxy terminus in the native hEGF does not affect the tertiary structure of the hEGF, and the structural stability, the host's stability to the protease, and the extracellular secretion of the hEGF as a fusion partner in the present invention. It does not affect the ease, recovery, purification and potency measurement. In addition, the plasmid vector pGBIle was cleaved with Sac I and Pac I restriction enzymes to obtain a 169 bp gene segment containing the hGRF gene, which was then cleaved with Mboll restriction enzymes to obtain a 138 bp hGRF gene segment. The 2917 bp fragment obtained from the expression vector pTE105 and the 138 bp hGRF gene fragment were conjugated with a 9 bp synthetic linker (see SEQ ID NOs: 3,4) containing methionine cleaved by CNBr and T4 DNA ligase to generate a new expression vector pEGRF. Is prepared (see FIG. 2).

Applicant deposited the transformant obtained by transforming the new expression vector pEGRF into Escherichia coli JM101 on March 27, 1997 to the Korean spawn association, which is an international depositing institution (Accession No .: KFCC-10963).

The gene sequence of the hGRF fused form of hEGF is as shown in SEQ ID NO: 1, the sequence of the hEGF-hGRF fusion protein expressed therefrom is shown in SEQ ID NO: 2.

Since the expressed hEGhGRF fusion protein is linked with an asparagine-methionine linker, it can be easily separated by CNBr, and the sequence of the finally obtained hGRF protein is shown in SEQ ID NO: 3.

Since the expression vector pEGRF of the present invention can express hEGF-hGRF fusion protein in E. coli and secrete it extracellularly, it is very suitable for efficiently producing hGRF protein. Specifically, the expression vector of the present invention uses a tac promoter (de Boer, et al ., DNA, 2, 231-235,1983; Amann, et al ., Gene, 25, 167-178, 1983) during protein expression. Expression of the hEGF-hGRF fusion protein allows easy control of its expression, and two ribosome binding sites (Shine and Dalgano, Pro. Natl. Acd. Sci, USA ,. 71, 1342, 1974) are located at the bottom of the promoter. ) The initiation of translation of the protein can be carried out more efficiently.

In addition, since the expression vector of the present invention contains an artificially synthesized ompA leader sequence, a universal translation stop sequence, and a trpA transcription stop sequence, the hEGF-hGRF fusion protein is easily and accurately expressed and secreted outside the cell. . In addition, the expression vector of the present invention contains a par site (Austin and Abeles, J. Mol. Biol., 169, 373-387, 1983), so that the vector can be stably maintained in E. coli.

Meanwhile, E. coli expression vectors generally use ampicillin-resistant markers, which are encoded by the genes encoded by beta lactamase to secrete them. Therefore, it is known that when the foreign protein is expressed and secreted extracellularly, it acts competitively with beta-lactamase to reduce secretion of the protein. Therefore, the expression vector of the present invention can be prepared to contain a tetracycline-resistant marker, the expressed hEGF-hGRF fusion protein can be effectively secreted extracellularly.

In addition, the overall size of the expression vector is very small, stable in the host cell and can efficiently express foreign proteins. In addition, the expressed hEGF-hGRF fusion protein can be easily separated and purified by CNBr treatment, and in this process, there is an advantage of simultaneously producing hEGF and hGRF.

In the amino acid sequence of the present invention, "functionally equivalent" means Gly, Ala in the amino acid sequence of the chimeric protein; Val, IIe, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And all proteins substituted with a combination such as Phe, Try. In addition, in the nucleotide sequence of the present invention, the term "functional equivalent" means all genes having a nucleotide sequence capable of providing the amino acid of the above-mentioned combination in the nucleotide sequence of the fusion gene.

Hereinafter, the present invention will be described in detail in the Examples.

The following examples illustrate the present invention in detail, and the content of the present invention is not limited by the examples.

Example 1 Preparation of Vector pGB11e Containing Gene Encoding [11e ²⁷ ] hGRF _1-44 Protein

In order to prepare the hGRF gene (Met2711e) designed in the present invention, after synthesizing oligonucleotide of 15mer consisting of 15 bases of SEQ ID NO: 3 and 23mers consisting of 23 bases of SEQ ID NO: 5, it contains the hGRF gene Cloning with plasmid vector pGB002. First, the plasmid vector pGBO02 600 nanogram was cut with Kpn 1 and Pst 1 restriction enzymes to separate 2952 bp DNA fragment of the plasmid vector portion. The same vector was digested with Bsm A1 and Kpn 1 restriction enzymes to isolate the 112 bp DNA fragment corresponding to the hGRF gene.

Completely dissolved in sterile secondary distilled water so that the concentration of each synthetic oligonucleotide is 100 picomoles per microliter in an Eppendorf test plate, and then 10 microliters of each oligonucleotide is added to the Eppendorf test tube. Mix and heat at 95 ° C. for 5 minutes and then slowly lower the temperature until the temperature reaches 37 ° C. and react.

100 nanograms of 112 bp DNA corresponding to the hGRF gene and 100 nanograms of the synthetic gene were added to 20 nanograms of the 2952 bp DNA fragment of the isolated plasmid vector and linked with 0.5 microliters of T4 DNA ligase. The vector was transformed into E. coli XL1-blue strain (Stratagere, Cat. No. 200249) by electroporation, and cultured in a medium containing 50 micrograms / milliliter of ampicillin to isolate a transformant.

The pBluescript 11 SK (+) plasmid vector in the transformant containing the hGRF gene thus prepared was named pGBlle, and subjected to 1% agarose gel electrophoresis after single and double cleavage with Kpn l, Sac l, Nla lll restriction enzymes. HGRF gene was correctly inserted and Met27lle substitution was confirmed.

Example 2 Preparation of Expression Vector pEGRF capable of Producing a Fusion Protein of hEGF and hGRF

In order to prepare an expression vector capable of producing a fusion protein of hEGF and hGRF in E. coli, the plasmid vector pGB1le containing the hGRF (Met271le) gene obtained in Example 1 was added to about 900 nanograms of restriction enzymes Sac l and Pac l 6, respectively. Microliters were added and reacted at 37 ° C. for 6 hours. After confirming cleavage by 1% agarose gel electrophoresis, cut the agarose gel in the region where the 169bp DNA fragment corresponding to the hGRF gene is located with a knife and use a QIAEX II DNA elution kit (Qiagen, Cat. No. 20021). Was dissolved in 20 microliter distilled water and separated purely. To this, 3 microliters of Mbo II restriction enzyme was further added and completely cleaved to obtain a 138 bp DNA fragment. Likewise, 3 microliters of restriction enzymes Afl ll and Pac l were added to about 500 nanograms of pTE105, an expression vector capable of producing hEGF protein, and reacted at 37 ° C. for 6 hours. The 2917 bp DNA fragment containing the plasmid vector and the amino terminus of hEGF was isolated and dissolved in 20 microliters of distilled water.

In order to introduce a methionine amino acid in front of the hGRF protein, a 14mer consisting of 14 bases of SEQ ID NO: 6 and a 9mer oligonucleotide consisting of 9 bases of SEQ ID NO: 7 were synthesized, and the concentration of each synthetic oligonucleotide was measured in an Eppendorf test tube. Completely dissolved in sterile secondary distilled water to make 100 picomoles per microliter. 10 microliters of each oligonucleotide was added to the Eppendorf test tube, mixed well, and then heated at 95 ° C. for 5 minutes, and then slowly lowered to a temperature of 37 ° C. for reaction.

2 microliters of 2917 bp expression vector containing hEGF digested with Afl ll, Pac l restriction enzyme, 6 microliters of 138 bp hGRF gene fragment digested with Pac l and Mbo 1I restriction enzyme, and 4 microliters of 9 bp synthetic gene After mixing well, 0.5 microliter of T4 DNA ligase was added and the reaction was carried out at 16 ° C. for 18 hours, followed by transformation into E. coli JM101 strain. An expression vector capable of producing a fusion protein of hEGF and hGRF through transformation was named pEGRF, and the hGRF gene was correctly inserted by single and double cleavage with Afl ll, Pac l, and Bst Xl restriction enzymes.

The transformant obtained by transforming the E. coli JM1O1 strain with the expression vector pEGRF of the present invention was deposited on March 27, 1997 to the Korean spawn association, which is an international depositing institution (Accession Number: KFCC-10963).

Example 3: Expression of E. coli transformant hEGF-hGRF fusion protein

To express the hEGF-hGRF fusion protein, E. coli strain JM101 transformed with the expression vector pEGRF was inoculated in 3 ml of LB medium containing tetracycline (12.5 μg / ml) and incubated at 30 ° C. for 18 hours. This culture was inoculated in 100 ml of the same medium such that the initial E. coli concentration was 0.1 (O.D) at a wavelength of 600 nm.

When shaken at 30 ° C and 250rpm, when E. coli concentration reached 0.5 (OD), IPTG (Sigma, Isopropy1-β-D-thiogalactopyranoside, Cat. No. 16758) was added to a final concentration of 1 mmol, hEGF- Expression of hGRF fusion protein was induced. After induction of expression, 1 ml of the culture solution was collected at 0, 1, 2, 4, and 6 hours, centrifuged at 12000 rpm for 3 minutes, and the supernatant and E. coli were separated and collected.

The protein in the periplasm was extracted from the isolated E. coli by the following method. To wash the collected E. coli, resuspend in phosphate buffered saline (pH7.4), centrifuge at 12000 rpm for 3 min at 4 ° C, and discard the supernatant. Add 10 times the volume of E. coli osmotic shock solution l (20 mM Tris-HC1, pH8, 2.5 mM EDTA, 20% Sucrose), resuspend and leave on ice for 10 minutes. Discard the supernatant after centrifugation at 4 ° C for 10 minutes at 8000 rpm. E. coli shock solution ll (20 mM Tris-HCI, pH8, 2.5 mM EDTA) of 10 times the volume of E. coli was added and then resuspended and left on ice for 10 minutes. The supernatant is collected after centrifugation at 4 ° C. for 5 minutes at 12000 rpm.

10 microliters of the culture supernatant and the ferricis protein samples were mixed with 10 microliters of reducing buffer solution (2 ×) for SDS-PAGE, and treated at 100 ° C. for 5 minutes, followed by SDS-PAGE. 3 (a) is the result of performing Coomassie staining after SDS-PAGE.

Lanes 1-5 are culture supernatants 0, 1, 2, 4 and 6 hours after expression induction, and lanes 6-10 are proteins in periplasm at 0, 1, 2, 4 and 6 hours after expression induction. .

Figure 3 (b) is the result of Western blot analysis of the same sample. Samples in each lane were the same as in the case of SDS-PAGE, and the primary antibody was used by diluting the antibody against hEGF made in rabbit in a phosphate buffer solution at a dilution ratio of 1: 1000, and the secondary antibody was a peroxidase. (Peroxidase) bound rabbit secondary antibody (Sigma, Cat. No. A0545) 릍 was used. It was confirmed that the hEGF-hGRF fusion protein is expressed at a basic level at a position corresponding to about 10,000 Daltons from the time of expression induction, and the expression amount is increased from 1 hour after expression induction. It was also confirmed that hEGF-hGRF fusion protein was secreted extracellularly after 4 hours of expression induction.

[Effects of the Invention]

The present invention suppresses the degradation of hGRF protein by intracellular protease by expressing hGRF protein in a large amount in the form of a fusion protein and secreting it extracellularly, thereby increasing the yield, and without destroying the cells when recovering hGRF protein. The hGRF protein can be recovered from the culture solution to prevent the incorporation of intracellular impurity proteins and to be isolated and purified.

Since the hEGF-hGRF fusion gene of the present invention expresses the hEGF-hGRF fusion protein, it is stably preserved in E. coli until hGRF is secreted extracellularly, and the expression vector pEGRF of the present invention allows the expression of the fusion protein in E. coli in large quantities. Since it can be secreted into the periplasmic region and extracellularly, the hGRF protein can be easily isolated and purified from the cell culture solution.

[Sequence list]

SEQ ID NO: 1

Sequence length: 279

Type of sequence: nucleic acid

Number of chains: 2 prints

Form: Straight

Type of sequence: DNA

[SEQ ID NO: 1]

AAC AGC GAC TCC GAA TGC CCG CTG AGC CAT GAC GGC TAC TGC CTG

TTG TCG CTG AGG CTT ACG GGC GAC TCG GTA CTG CCG ATG ACG GAC

CAC GAC GGC 印 A TGC AT knee AC ATC GM GCA CTG GAC AAA TAC GCG

GTG CTG CCG CAT ACG TAC ATG TAG CTT CGT GAC CTG TTT ATG CGC

GAC CTT MC ATG TAC GCT GAC GCT ATC TTC ACT MC TCT TAC CGT

CTG GAA TTG TAC ATG CGA CTG CGA TAG AAG TGA TTG AGA ATG GCA

AAA GTT CTG GGT CAG CTG TCT GCT CGT AAA CTG CTG CAG GAT ATC

TTT CM GAC CCA GTC GAC AGA CGA GCA TTT GAC GAC GTC CTA TAG

ATC TCT CGT GAG CAG GGT GAA TCT AAC CAG GAA CGT GGT GCA CGT

TAG AGA GCA CTC GTC CCA CTT AGA TTG GTC CTT GCA CCA CGT GCA

GCG CGC CTG

CGC GCG GAC

[Sequence list]

SEQ ID NO: 2

Sequence length: 93

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Peptide

[SEQ ID NO: 2]

Asn Ser Asp Ser G1u Cys Pro Leu Ser His Asp Gly Tyr Cys Leu

His Asp G∥y Val Cys Met Tyr lle Glu Ala Leu Asp Lys Tyr Ala

Cys Asn Cys Val Val Gly Tyr group Gly G∥u Arg Cys G1n Tyr Arg

Asp Leu Asn Met Tyr Ala Asp Ala lle Phe Thr Asn Ser Tyr Arg

lle Ser Arg Glu Gln Gly Glu Ser Asn Gln Glu Arg Gly A1a Arg

Ala Arg Leu

[West List]

SEQ ID NO: 3

Sequence length: 44

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Hittide

[SEQ ID NO: 3]

Tyr Ala Asp Ala lle Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly

Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp I1e lle Ser Arg Glu

Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu

[Sequence list]

SEQ ID NO: 4

Sequence length: 14

Type of sequence: nucleic acid

Number of sand: 1 print

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 4]

GATATCATCT CTCG

[West List]

SEQ ID NO: 5

Sequence length: 23

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 5]

CTGACGAGAG ATGATATCCT GCA

[Sequence list]

SEQ ID NO: 6

Sequence length: 14

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 6]

TTMCATGTA CGCT

[West List]

SEQ ID NO: 7

Sequence length: 9

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 7]

GCGTACATG

Claims (13)

A human epidermal growth factor-human growth hormone-releasing factor fusion gene linked to a human epidermal growth factor (hEGF) gene and a human growth hormone release factor (hGRF) gene substituted with isoleucine instead of the methionine amino acid No. 27. The human epidermal growth factor-human growth hormone releasing factor fusion gene of claim 1, wherein the human epidermal growth factor (hEGF) gene and the human growth hormone releasing factor (hGRF) gene are linked by a linker gene encoding asparagine-methionine. The human epidermal growth factor-human growth hormone releasing factor fusion gene according to claim 1 or 2, wherein the gene has a nucleotide sequence of SEQ ID NO: 1. A plasmid expression vector pEGRF containing the human epidermal growth factor-human growth hormone-releasing factor fusion gene of claim 3. The plasmid expression vector pEGRF according to claim 4, wherein the expression of the human epidermal growth factor-human growth hormone releasing factor fusion gene is regulated by the tac promoter. The plasmid expression vector pEGRF according to claim 4, wherein the epidermal growth factor-human growth hormone-releasing factor fusion protein can be expressed by the omp A leader sequence and secreted into the cell culture medium. A transformant obtained by transforming E. coli JM101 strain (Escherichia coli JM101) with the plasmid expression vector pEGRF of claim 4 (Accession Number: KFCC-10963). Human epidermal growth factor-human growth hormone-releasing factor fusion protein expressed from the human epidermal growth factor-human growth hormone-releasing factor fusion gene of claim 1 9. The human epidermal growth factor-human growth hormone releasing factor fusion protein of claim 8, wherein the amino acid sequence of the fusion protein is SEQ ID NO: 2. A method of producing a human growth hormone-releasing factor protein by culturing the host microorganism transformed with the recombinant DNA of claim 1, obtaining a fusion protein in which human epidermal growth factor and human growth hormone-releasing factor are fused, and cutting the fusion protein. . The method of claim 10, wherein the human growth hormone release factor protein has a amino acid sequence represented by SEQ ID NO: 3. The method of claim 10, wherein the fusion protein is cleaved using cyanobromide (CNBr) to prepare human growth hormone releasing factor protein. A method for producing human growth hormone release factor by culturing the host microorganism transformed with the recombinant DNA of claim 1, obtaining a fusion protein in which human epidermal growth factor and human growth hormone release factor are fused, and cleaving the fusion protein Human growth hormone release factor protein prepared by