KR100295909B1 - Vector including human epidermal growth factor(egf) and manufacturing method of egf by using the same - Google Patents

Abstract

PURPOSE: Provided are a vector including human epidermal growth factor(EGF) which promotes the growth of human epidermal cells and inhibits the secretion of gastric acid and a manufacturing method of EGF by using the vector. CONSTITUTION: The vector including EGF is manufactured by the steps of: inserting T-mox(MOX terminator) into a recognition site of pUC19 vector to prepare pUC-Tmox vector; inserting LEU2 gene into a recognition site of pUC-Tmox to prepare pUC-TaLE vector; inserting P-mox gene(MOX promotor), alpha pre-proleader(ppL) sequence and EGF gene into a recognition site of SalI of pUC-TaLE vector; cutting pU-EGF2 vector with EcoRI restriction enzyme; and inserting a fragment including P-mox/ppL/hEGF/T-mox into a recognition site of EcoRI of aP3 vector.

Description

A vector having a gene of human epidermal growth factor and a method of producing human epidermal growth factor using the same

1 is a diagram showing an embodiment of the DNA sequence and amino acid sequence of the synthetic hEGF gene of the present invention,

Figure 2 is an embodiment of a vector comprising the hEGF of the present invention and its manufacturing process,

Figure 3a is a photograph showing a portion of the result of the orientation of the Hansenula polymorpha transformed with a vector containing hEGF, an embodiment of the present invention on a nitrocellulose disk,

Figure 3b is a photograph showing a part of the results of the immunoassay of Hanshenula polymorpha transformed with a vector containing hEGF of one embodiment of the present invention on a nitrocellulose disk,

4 is a photograph showing the results of SDS-PAGE analysis of hEGF produced by the present invention and hEGF produced by other bacteria,

Figure 5a is a photograph showing the results of the SDS-PAGE analysis of the hEGF produced according to the present invention,

Figure 5b is a photograph showing the result of Western blotting of hEGF produced according to the present invention,

Figure 6a is a photograph showing the result of Southern blotting by treatment with Hansenula polymorpha transformed according to the invention Hind III,

Figure 6b is a photograph showing the result of Southern blotting by treatment with Hansenula polymorpha transformed according to the present invention EcoR I,

7 is a photograph showing the results of Southern blotting of a promoter in Hanshenula polymorpha transformed according to the present invention.

[Commercial Use]

The present invention relates to epidermal growth factor (EGF), and more particularly to a recombinant vector comprising a gene of epidermal growth factor of the human body and a method for producing a large amount of epidermal growth factor of the human body using the same. will be.

[Prior art]

Epidermal growth factor (EGF) is a relatively small 6KDa short-chain polypeptide consisting of 53 amino acids that stimulates the growth of epithelial cells and fibroblastic cells (Carpenter, G., 1978, The regulation of the cell proliferation: Advances in the biology and mechanism of action of epidermal growth factor.J. Invest.Dermatol. 71: 283-287), ulcer suppression (Gregory, II., 1975, Isolation and structure of urogastrone and its relationship to epidermal growth factor, Nature 257: 325-327). Such EGF has been researched and developed for therapeutic uses such as wound or burn treatment, corneal superficial wound treatment, corneal transplant surgery, and gastric ulcer treatment using the properties described above.

However, despite the biological and pharmacological significance as described above, these EGF has a problem that only a very small amount can be extracted from the animal cells and mass production is impossible. In order to solve this problem, studies have been attempted to mass-produce EGF by genetic recombination technology. Representative genetic recombination technology for mass production of EGF studied so far is to introduce synthetic human EGF gene into Escherichia coli and express it (Fumitaka, K., G. Hideyki, K. Masaharu, N). Tomoyuki, K. Tomoko, A. Hideo, S. Seizo and O. shigeo, 1986, Direct expression of urogastrone gene in Esherichia coli, Gene, 45: 311-316), introduced into and expressed by Bacillus brevis. Method (Hideo Y., N. Kazuo, S. Yutkaka, and K. Atsushi, 1989, Use of Bacillus brevis, for efficient synthesis and secretion of human epidermal growth factor, Proc. Nat 1. Acad. Sci. USA 86: 3589- 3593). However, as described above, the method of expressing the human EGF gene, which is a gene of higher eukaryotic cells, using a prokaryotic bacterium as a host, does not allow the prokaryotic RNA polymerase to recognize the promoter of the eukaryotic gene or the eukaryotic gene. Proliferation of human EGF is difficult due to the lack of prokaryotic ribosomes, post-translational processing of the resulting proteins, or degradation of these proteins by prokaryotic proteases. have.

In order to solve the problems caused when using such prokaryotic cells as a host, the method of intracellular expression using Saccharomyces cerevisiae as a host in yeast, a lower eukaryotic cell (Ureda, MA, JP Merryweather) , GT Mullenbach, D. Coit, U. Heberlein, P. Valenzuela and PJ Barr. 1983, Chemical synthesis of a genen for human epidermal factor urogastrone and its espression in yeast, Proc. Natl. Acad. Sci. USA 80: 7461- 7465), or a method of effectively secreting in vitro by using a pre-pro leader sequence derived from an α-factor that is a cross pheromone (Brake, AJ, JP Merryweather, DG Coit, UA Heberlein, FR). Masiarz, GT Mullenbach, MS Uredea, P. Valenzuela, and PJ Barr, 1984, α-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. USA, 81: 4642-4646) . This method of expressing heterologous proteins using Saccharomyces cerevisiae has high productivity and shows a general post-translational process of higher eukaryotic cells. It is used. However, Saccharomyces cerevisiae does not have a powerful and finely regulated promoter for mass production of heterologous proteins in terms of industrial applications, and organic culture is required for high concentration culture. Heterologous proteins are highly glycosylated and are not suitable as host cells (Romanos, MA, CA Scorer and JJ Clare, 1992, Foerign gene expression in yeast: a review, Yeast 8: 423-488) .

Recently, in order to supplement the above disadvantages of Saccharomyces cerevisiae, a method of secreting rat EGF using methanol and yeast Pichia pastoris (Clare, JJ, MA) has been studied. Romanos, and FB Rayment, 1991, Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies, Gene, 105: 205-212).

However, in order to produce EGF, Pichia pastoris has to be cultured in a medium for mass production containing glycerol after being incubated for a certain period in a medium containing methanol to produce EGF. There is a problem that must be incubated in stages.

Recently, research on the development of heterologous protein expression systems using Hansenula polymorpha, another metanol magnetization yeast, has been actively conducted (Gellissen, G., ZA Janowicz, A. Merckelbach, M. Piontek, P.). Keup, U. Weydemann, CP Hollenberg and AWM Strasser, 1991, Heterologous gene expression in Hansehula polymorpha: efficient Secretion of glucoamylase, BIO / TECHNOLOGY, 9: 291-195). Methanol magnetization yeast Hanshenula polymorpha has high economic feasibility by using methanol as energy source and carbon source, and has strong and relatively easy to regulate promoter such as promoter of methanol oxidase (MOX) gene. This is high. In addition, high concentration culture (100g DCW / L) is easy and the expression target genes are introduced several times on the host chromosome during transformation can increase the amount of foreign protein expression. In addition, the stability of the plasmid introduced in the non-selective medium is excellent, and high glycosylation does not generally occur when the foreign protein is expressed. In addition, EGF can be produced by only one step culture on a medium containing glycerol or glycerol / methanol.

[Purpose of invention]

The present inventors continue to study and express human EGF using Hansenula polymorpha, a metanol-magnetizing yeast having the above-mentioned advantages, as a host cell, and thus, it is suitable for expressing human EGF in Hansenula polymorpha. Vectors were prepared and expressed in human EGF using recombinant Hansenula polymorpha transformed with the vector.

The present invention has been made to solve the problems of the prior art as described above, an object of the present invention is to provide a vector capable of effectively mass-producing human EGF using the first Hansenula polymorpha as a host cell, and the second vector The present invention provides a transformant capable of effectively mass-producing human EGF introduced into Hanshenula polymorpha, and thirdly, a method of effectively mass-producing human EGF using such transformant.

[Configuration of Invention]

The present invention provides a pU-EGF2 recombinant vector comprising a MOX promoter-ppL (α pre-pro leader) -hEGF-MOX terminator gene located between pUC19 and EcoRI and SalI recognition sites of pUC19.

The present invention also provides a pUC-Tmox vector by inserting a T-mox (MOX terminator) into the BamHI / SmaI recognition site of the pUC19 vector, and inserting the LEU2 gene into the SalI recognition site of the pUC-Tmox vector to the pUC-TaLE vector. Prepare a pU-EGF2 vector by inserting the P-mox gene (MOX promotor), α pre-proleader (ppL) sequence, and the EGF gene into the SalI recognition site of the pUC-TaLE vector, The pU-EGF2 vector is cut with an EcoRI restriction enzyme, and a pA-EGF3 recombinant vector prepared by inserting a fragment containing P-mox / ppL / hEGF / T-mox into an EcoRI recognition site of the pA3 vector is provided.

MOX is methanol oxidase, hEGF is human epidermal growth factor (EGF).

And the present invention provides a Hansenula polymorpha transformant transformed with the pA-EGF3 recombinant vector described above.

The transgenic strain of the present invention was deposited with the Korea Institute of Science and Technology Gene Bank (KCTC) on May 26, 1994 under the name Hanshenula polymorpha 211, and the accession number is KCTC 8601P. The above-mentioned Hansenula polymorpha is preferably Hansenula polymorpha DL-1.

In another aspect, the present invention provides a method for producing human EGF using the Hansenula polymorpha transformant.

Also provided is a recombinant human EGF protein of the invention. The recombinant human EGF protein can be expressed extracellularly to yield up to 24 mg / L.

EXAMPLE

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only preferred embodiments of the present invention to aid in understanding the present invention, and the present invention is not limited to the following examples.

I. Materials and Methods

1. Used strains and plasmids

Hansenula polymorpha DL-1 (leu2), A16 (leu2), and HPB1 (ade2 leu2) were used as the methol magnetization yeast strain, and Escherichia coli DH5α (F lacZAM15 hsdR17 (FlacZAM15 hsdR17) was used for general DNA manipulation and plasmid isolation. r- m-) gyrA36 was used.

Plasmids include pMOX7ΔRI (Sohn, JH, MY Beburov, ES Choi, and SK Rhee, 1993, Heterologus genen expression and secretion of the anticoagulant hirudin in a methylotrophic yeast Hansenula polymorpha, containing a MOX promoter and a MOX terminator). PA3 containing J. Micorbio L. bIOTECHNOL, 3: 65-72) and the LEU2 gene of Hanshenula polymorpha and Autonomously Replicating Sequences of Hansenula (HARs) Aliquots were used from Ter-Avanesyan (Cardiology Research Center, Russia). PUC19 (Yanisch-Perron, C., J. Vieira, and J. Messing, 1989, Molecular Cloning, A Laboratory Manual, 2nd ed.) For the preparation of vectors for introducing foreign genes into the chromosomal DNA of Hanshenula polymorpha. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA).

2. Use badge

Nutrient medium used for cultivation of Methanol magnetized yeast was YPD (yeast extract 1%, bacto-peptone 2%, dextrose 2%) medium and YPD agar (YPD broth, agar 2%) medium. As a medium for culturing the transformed transformant, a medium having a composition of YNB (yeast nitrogen base w / o amino acid) 0.67% and dextrose 2% was used. As an expression induction medium for measuring the activity of foreign proteins in transformed yeast, a medium having a composition of YNB 0.67%, glycerol 0.5%, methanol 1%, and casamino acid 1% − was used. In addition, as a medium for inducing colonies on the nitrocellulose disk, yeast extract 1%, bacto-peptone 2%, methanol 1%, agar 2 Agar induction medium of% composition was used.

3. Restriction Enzymes and Reagents

Restriction enzymes for DNA recombination, T4 DNA ligase, calf-intestine alkaline phosphatase, RNase and the like were purchased from Boehringer Mannheim. ³⁵ S-dATPαS from Dupont, mouse anti-hEGF antibldy from Pierce, Alkaline Phosphatase (AP) labeled anti-mouse IgG Was purchased from Bio-Rad, and anti-mouse IgG (HRP) labeled with HRP (Horse radish peroxidase) was purchased from Vector, respectively. Reagents and antibiotics used in buffers , X-Gal and standard recombinant human EGF were purchased from Sigma. Separation and purification of DNA from Agarose gel was used by purchasing GENECLEAN II and MERMAID kit from Bio101.

4. How to

DNA recombination process in the following examples are described in Sambrook (J. Brook, J., EF Fritsch, and T. Maniatis, 1989, Molecular Cloning, A laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA), and Escherichia coli transformation was carried out in Inoue (H., H. Nojima, and H. Okayama, 1990. High efficiency transformation of Escherichia coli with plasmids, Gene, 96: 23-28). Transformation and isolation of DNA from Hanshenula polymorpha were performed by Sohn, JH, MY Beburov, ES Choi, and SK Rhee, 1993, Heterologus genen expression and secretion of the anticoagulant hirudin in a methylotrophic yeast Hansenula polymorpha, J. Micorbiol. Biotechnol, 3: 65-72).

II. Human EGF Gene Synthesis

Amino acid sequences of known human EGF (Harper, RA, J. Pierce, and CR Savage, Jr. 1987, Purification of human epidermal growth factor by monoclonal antibody affinity chromatography, p. 3-11, In D. Barnes and DA Sirbasku, Methods In Ezymol., Vol. 146, Academic Press, New York, London), the human EGF gene sequence was determined according to the codon usage of the Hansenula polymorpha MOX gene, and accordingly the human EGF gene was chemically synthesized. This will be described in more detail as follows.

As shown in FIG. 1, in order to synthesize the human EGF gene, 10 overlapping oligonucleotides S1 to S10 whose ends were 33-43mer in length were complementary to each other. Synthesis and purification of the oligonucleotides were performed by Korea Biotech. Then, the separated and purified oligonucleotides were annealed and ligated using the same molar concentration to complete the human EGF gene. At this time, the annealing and ligation of each purified oligonium nucleotide is performed by Sohn, JH, SK Lee, ES Choi and SK Rhee, 1991, Gene expression and secretion of the anticoagulant hirudin in Saccharomyces cerevisiae, J. Microbiol Biotechnol. 1: 266-273). Human EGF gene thus synthesized was 0.8% agarose gel electrophoresis to cut the DNA band of the appropriate size was isolated human EGF gene using the MERMAID kit. In addition, the 5 'end of the isolated human EGF gene is recognized by KEX2 endopeptidase of a cross-linking factor α, and the DNA sequence corresponding to Lys-Arg and restriction enzyme XbaI can be precisely processed. An adapter DNA sequence (dCGCCGTCTAGATAAGAGA) with a recognition site was introduced, and a SalI recognition site was introduced at the 3 'end. The gene thus synthesized was cut into XbaI and SalI, introduced into the XbaI-SalI site of pBluescript KS +, and subcloned to bindide chain-termination (Sanger, F., S. Nicklen, and AR Coulson). , 1977, DNA seauencing with chain terminating inhibitors, Porc. Natl. Acad. Sci. USA, 74: 5463-5467) showed that the human EGF gene was completely synthesized.

III. Expression and Secretion Vector pA-EGF3 Preparation

In order to express and secrete human EGF using Hansenula polymorpha, as shown in FIG. 2, a fragment of MOX terminator (T-mox) was isolated from pMOX7ΔRI and recombined with pUC19 to prepare pUC-Tmox. The LEU2 gene, SalI-XhoI fragment, was isolated from YEp13 and recombined into pUC-Tmox to prepare pUC-TaLE. In addition, these fragments from pBKS-K3, which were recombined with the correct interlinking of the α-pre-pro leader (ppL) sequence, a secretion signal sequence derived from the MOX promoter (P-mox) and Saccharomyces cerevisiae Was isolated and fragments of human EGF gene were isolated from pBEGF1 where the synthesized human EGF gene was recombined. PU-EGF2 was prepared by recombining the MOX promoter-ppL-hEGF fragments so linked to pUC-TaLE in order. And the expression and secretion cassette of the MOX promoter-ppL-hEGF-MOX terminator isolated from pU-EGF2 was isolated and recombined into a pA3 plasmid containing Hansenula polymorpha LEU2 gene and HARS to prepare pA-EGF3.

IV. Selection of human EGF-secreting transformants

Five days after pA-EGF3 was transformed into several kinds of Hanshenula polymorpha, approximately 2000 colonies on the selection medium were picked with toothpicks on YPD agar medium and incubated at 37 ° C for 24 hours ( See also FIG. 3a) Replicate to nitrocellulose disks and transfer cultured for 20 hours at 37 ° C in agar induction medium containing metaol. Colony-attached nitrocellulose discs were washed with 20 mM Tris-HCI (pH 7.5) / 500 mM NaCl and treated with 5% (W / V) skim milk powder and treated with TBST (10 mM Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4). Rat anti hEGF antibodies were diluted in TBS (10 mM Tris, 0.15 M NaCl, pH 7.4) and then treated with nitrocellulose discs for 1 hour. This was again washed with TBST, treated with AP-labeled anti-rat IgG for 1 hour, washed and developed by NBT (p-nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-indoyl phosphate). Colonies on YPD corresponding to were selected. The immunoassay results as shown in Figure 3b.

As shown in FIG. 3b, the degree of color development was different for each transformant. Among them, 240 strains of human EGF-secreting strains showing a strong signal were first selected, and each strain was expressed in an expression-inducing medium, and the concentration of human EGF in the culture supernatant was measured by ELISA. Seemed. The results in Table 1 are the same as the immunoassay results on the nitrocellulose discs, and it is thought that the different amounts of human EGF are secreted from each strain because the degree of introduction of the human EGF gene into the chromosomal DNA of the host cell is different. As a result of ELISA measurement of the culture supernatant of approximately 240 strains, DL-1 showed the highest human EGF production rate among the three Hansenula polymorpha strains (colonies No. 2-21, 24mg / L). Compared with the strains (HPB1 and A16) it was determined to be suitable as a human EGF production strain because of the high resistance to methanol and the rapid growth rate of the cells.

TABLE 1

V. Confirmation of Preparative Human EGF

To confirm the protein pattern in the culture medium of human EGF-producing strains, DL-1 (second lane) and DL-1 (third lane) having vector pA3 were used as controls, and pA-EGF3 transformed into human EGF secreting strain. 4 strains (lanes 4 to 8) containing the DL-1 strain and Saccharomyces cerevisiae strain (lane 9), also identified as a human EGF secretion strain, were incubated for 60 hours in an expression-inducing medium. . These culture supernatants were treated with TCA to obtain proteins and SDS-PAGE to confirm their molecular weight. As shown in FIG. 4, recombinant human EGF and Saccharomyces expressed in the strain containing pA-EGF3. Recombinant human EGF expressed in cerevisiae showed the same pattern.

In addition, the transformants transformed by the plasmid containing the secretion signal among these DL-1 strains were incubated for 24 hours in an induction medium, followed by incubation for 24 hours again with the addition of 1% of methanol, followed by centrifugation. After adding 50% TCA (trichloroacetic acid) to the culture supernatant, the mixture was left at 0 ° C for 1 minute and then centrifuged at 10000 rpm for 10 minutes to wash the remaining TCA with a mixture of ether and ethanol 1: 1. Dried over. This was followed by SDS-PAGE (see also 5a) according to the Laemli method (Laemmli, UK, 1970, Cleavage of structure proteins during the assembly of the head bacterophage T4, Nature, 227: 680-685) and human EGF specific monoclones. Western blotting using raw antibodies (see also Figure 5b).

As can be seen in Figure 5b it was confirmed that recombinant human EGF expressed in Hanshenula polymorpha has the same epitope as the human EGF derived from urine.

VI. Confirmation of the introduction of the vector pA-EGF3 into the Hanshenula polymorpha chromosome

To confirm the introduction of pA-EGF3 into the Hansenula polymorpha chromosome, pA-EGF3 (lane 1), pA-EGF3 and whole DNA (lane 8) of the DL-1 host, and the total DNA of the DL-1 host (Lane 13) and strains secreting human EGF 1-7 (lane 2) 1-16 (lane 3) 1-71 (lane 4) 2-11 (lane 5) of 10 strains (Table 1) ) 2-21 (lane 6) 2-23 (lane 7) 2-76 (lane 9) 3-6 (lane 10) 3-11 (lane 11) 3-25 (lane 12) The whole DNA was treated with HindIII (see also FIG. 6a) and EcoRI (see also FIG. 6b), electrophoresed on an agarose gel and then transferred to a nylon membrane filter, followed by Southern hybridization according to Samburg et al. (Southern hybridization) was carried out, the pA-EGF3 treated with HindIII as a probe was used by labeling according to the instructions using a DIG labeling kit (manufactured by Schöllinger Mannheim). The results are shown in FIG.

As can be seen in Figure 6 it can be confirmed that the plasmid-derived gene is present in the chromosomal DNA of all transformants showing EGF activity. For 1-7, 1-71, 2-21, 2-76, 3-6,3-25 weeks, the 9.2 kb pA-EGF3 / HindIII fragment used as a control in Figure 6a and pA-EGF3 in Figure 6b. A band showing a stronger signal than the other bands was observed at the same position as the / EcoRI sections 5.8 and 3.4 kb, respectively. These are aspects in which at least two plasmid DNAs are introduced as tandem-repeat into the chromosome of a host cell, in particular colony No. In lane 6 corresponding to 2-21, the difference in relative signal strength was greater than that in other lanes. This shows that the number of plasmids introduced into tandem-repeat is the highest and is in agreement with the previous experimental results with the highest human EGF productivity of the same strain.

But colony no. As in the case of 2-11, the form of mass introduction was not seen, but a high-productivity strain of human EGF was observed.

Iii. Confirmation of the number of clones introduced into the vector pA-EGF3

In order to confirm the number of gene copies derived from plasmids of each transformant, MOA promoter fragments obtained by treating StuI / EcoRI with pA-EGF3 and pMOXΔRI were DIG-labeled and used as probes. The result is also shown in FIG.

As shown in FIG. 7, the signal corresponding to the host's MOX promoter fragment appeared near 0.8 Kb and the signal corresponding to the DNA from plasmid pA-EGF3 appeared near 1.6 Kb. The difference in signal strength of these two bands is due to the numerical difference between the single copy of the MOX promoter fragment originally possessed by the host Hanshenula polymorpha DL-1 and the MOX promoter fragment derived from pA-EGF3 transformed. The number of copies of the plasmid pA-EGF3 introduced into the chromosomal DNA of the converter was confirmed. As shown in the results, colony no. At 1-7, 1-16, 2-21, 2-76, 3-6, 3-11, and 3-25, signals of 1.6 and 0.8 kb appeared as expected. Comparing these two signal strengths showed a difference of about 2-5 times and colony no. In case of 2-21, the difference in signal intensity was found to be about 5 copies introduced into the chromosome. And No. 1-71, 2-11, and 2-23 strains showed no relative bands to compare, which were introduced around the MOX genes on the chromosome when the plasmid genes were introduced into the chromosomal DNA, altering their hybridization patterns. It was estimated.

Iii. Standard Curve of Human EGF

The primary selected human EGF secretion strains were shaken in vitro for 24 hours at 37 ° C. in a test tube containing expression induction medium, followed by shaking at 37 ° C. for 24 hours with the addition of 1% methanol. The culture solution was diluted 100-fold with PBS (0.008 M Na ₂ HPO ₄ , 0.002 M KH ₂ PO ₄ , 0.14 M NaCl, 0.01 M KCl, Ph 7.4), and then 100 μl of the dilution was placed in a microtiter well (Nunc-immuno module). Each was added and reacted at 37 ° C for 1 hour. It was washed with TBST, treated with mouse anti-hEGF antibody for 1 hour, and washed again with TBST three times. This was treated with HRP-labeled anti-rat IgG for 1 hour, washed, and mixed with ABTS (2,2'-azino-di- [3-ethylbenzthiazoline sulfonate (6)], H ₂ O ₂ in a 1: 1 mixture. Colors were added by adding μl and diluted to 50 ng of standard human EGF at 405 nm using a 96-well plate auto reader (THERMOmax, Molecular devices, USA) to prepare a standard curve.

IX. Production of Human EGF

After inoculating human EGF into a culture medium containing 50 g of yeast extract, 100 g of bacto-peptone, 100 g of glycerol, and 2 l of distilled water using the DL-1 strain, and incubating at 37 ° C., YNB (Yeast Nitrogen Base Without amino acid) 67 g, glycerol 1000 g, methanol 1000 g, was incubated while maintaining the methanol 0.5-1% by weight in an induction medium containing 2 L of distilled water. About 50 hours after induction, the concentration of human EGF was 200 mg / l.

[effect]

Human EGF recombinant vector of the present invention contains a MOX promoter is easy to control the production of human EGF, contains HARS, has a high transformation rate, and has a pre-proderer to facilitate protein secretion. In addition, the Hansenula polymorpha is a eukaryotic cell gene and can stably produce human EGF, and can include human EGF vectors of the present invention in large quantities. It is economical to use.

Claims (4)

A pUC-Tmox vector was prepared by inserting a T-mox (MOX terminator) into the BamHI / SmaI recognition site of the pUC19 vector, a pUC-TaLE vector was prepared by inserting the LEU2 gene into the SalI recognition site of the pUC-Tmox vector, A pU-EGF2 vector was prepared by inserting a P-mox gene (MOX promotor), an α pre-proleader (ppL) sequence and an EGF gene into the SalI recognition region of the pUC-TaLE vector, and the pU-EGF2. PA-EGF3 recombinant vector prepared by cutting the vector with EcoRI restriction enzyme and inserting a fragment containing P-mox / ppL / hEGF / T-mox into the EcoRI recognition site of the pA3 vector. The Accession No. KCTC 8601P Hansenula polymorpha transformant transformed with the pA-EGF3 recombinant vector of claim 2. The Hansenula polymorpha strain of claim 2, wherein the Hansenula polymorpha is Hansenula polymorpha DL-1. A method of producing human EGF by culturing the Hansenula polymorpha transformant of claim 3.