KR100260633B1 - Expression vector of human midkine gene - Google Patents

Abstract

PURPOSE: An expression vector containing the human midkine gene, a recombinant yeast transformed by the expression vector, and a method for producing the human midkine protein using the recombinant yeast are provided, thereby the midkine can be mass produced, so that it can be used in the treatment of Alzheimer's disease and Parkinson's disease. CONSTITUTION: Midkine is a gene relating to cell differentiation. The method for producing the human midkine protein using the recombinant yeast comprises the steps of: preparing primers and amplifying the human midkine gene by PCR with cDNA of human fetal brain as a template using primers; ligating the ubiquitin gene and the amplified human midkine gene with ligase; inserting the fusion gene into a plasmid pYLBC-A/G-UB-HGH to produce a plasmid pYLBC-A/G-UB/MK; transforming Saccharomyces cerevisiae with the plasmid pYLBC-A/G-UB/MK to produce Saccharomyces cerevisiae DC04-UM-MK, pYLBC-A/G-UB/MK(ATCC 74259); and incubating the recombinant Saccharomyces cerevisiae DC04-UM-MK, pYLBC-A/G-UB/MK(ATCC 74259) in an appropriate medium to produce the human midkine protein.

Description

Expression Vectors of the Human Midkine Gene and Its Expression in Yeast

1 shows the amino acid sequence of human midkine,

2 shows the connection strategy of the human midkine gene and the manufacturing process of the yeast expression vector,

Figure 3 shows the results of SDS-polyacrylamide gel electrophoresis after the expression of human midkine using the human midkine expression vector (pYLBC-A / G-UB-MK) of the present invention.

The present invention relates to an expression vector for the production of a human midkine protein, a yeast transformed with the expression vector, and a method for producing human midkine using the same. A human midkine expression vector comprising a human midkine gene fused to a ubiquitin gene, which can be used to produce midkine in large quantities, and a yeast transformed with the expression vector and a protein produced thereby.

In general, bioactive factors or growth factors are known to play an important role in the regulation of cell growth and cell differentiation (Slack, JM et al., Nature 326, 197-200 (1987); Rosa, F. et al. , Science 239, 783-785 (1988); Murata, M. et al., PNAS 85, 2434-2438 (1988); Thomas, KR et al., Nature 346, 847-850 (1990); McMahon, AP et al., Cell 62, 1073-1085 (1990)), which belong to the fibroblast growth factor (basic FGF), acidic fibroblast growth factor (acidic FGF), int- Several growth factors are known, such as 2 and hst, and they are also called heparin binding growth factors because they have heparin binding capacity.

Recently, new heparin-binding growth factors have been reported from several groups that show structurally significant differences from these fibroblast growth factors, for example, heparin binding neurotrophic growth-associated molecule (HB-GAM: Merenmies, J. et al., J. Biol. Chem. 265, 16721 (1990)), pleiotrophin (PTN: Li, YS et al., Science 250, 1690 (1990)), heparin binding nerve growth factor (heparin binding neurotrophic factor, HBNF: Kovesdi, J. et al., Biochem. Biophys. Res. Commun. 172, 850 (1990)), and midking (MK: Kadomatsu, K. et al., Biochem. Biophys Res. Commun. 151, 1312 (1988)).

The midkine was first identified by the Katomatsu group in the early stages of differentiation induced by retinoic acid in culturing mouse embrional carcinoma cells (EC). In other words, they are derived from the fact that fetal carcinoma (EC) cells remain undifferentiated under appropriately controlled conditions and that differentiation is induced by retinoic acid (McBurney, MW et al., Nature 299, 165 (1982)). It was estimated that there was a gene involved in the differentiation process, and cloned the gene expressed early in the differentiation (Kadomatsu, K. et al., BBRC 151, 1312-1318 (1988)), and this gene was on the fifth day of gestation in rats. Was found throughout the fetus on days 7-9 and only in the kidney on day 15 (Kadomatsu, K. et al., J. Cell. Biol. 110, 607-616 (1990)). . Therefore, it was named midkine because it was found in the kidney of midgestion.

Tomomura et al. Identified three types of cDNA that differ in the 5´-untranslated region (MK1, MK2, MK3), of which MK2 is caused by retinoic acid in fetal carcinoma cells. It was found in the major midkine RNA that is derived (Tomomura, M. et. Al., J. Biol. Chem. 265, 10765 (1990)). In addition, Tsutsui et al. Isolated human midkine cDNA from human fetal kidney cDNA library using mouse cDNA as a probe, and compared the amino acid sequence of mouse and human midkine. (Tsutsui, J. et al., BBRC 176, 792-797 (1991)).

On the other hand, Tomomura et al. Showed that heparin-binding ability of both the midkine protein secreted early in the differentiation of mouse fetal carcinoma cells and the midkine protein secreted from fibroblast L cells transformed with the mouse midkine gene. It has been reported that it is a factor involved in the regulation of cell growth and differentiation by promoting the growth of PC12 cells (Tomomura, M. et al., BBRC 171, 603-609 (1990)).

A comparison of the amino acid sequences of heparin-bound neuronal growth factor (HBNF) and midcaine showed approximately 55% similarity, and the positions of nine cysteine residues matched, suggesting similarity of the tertiary structure of the two substances. And midkine, similar to HBNF, which already has neurostimulating activity, was thought to have similar biological activity (Kovesdi I. et al., BBRC 172, 850-854 (1990)). In addition, similarities between heparin binding growth-related substances / playotropin and midkine sequences have been reported to be quite high (Tsutsui et al., BBRC 176, 792-797 (1991); Naito et al., BBRC 183, 701-). 707 (1992).

Kovesdi et al. Obtained the midkine gene from the cDNA library obtained from the human brain, expressed it in E. coli, purified the midkine protein using heparin affinity column and ion exchange resin, and measured the biological activity. Reported nearly the same activity as (EP 476233 (1991)). Recently, Muramatsu et al. Reported the activity after pure purification of recombinant midkine secreted from L cells by heparin affinity column and hydrophobic chromatography (Biochem. Biophys. Res. Commun. 177, 652-658 ( 1991).

However, the E. coli expression system has a problem that it is difficult to remove the E. coli-derived exothermic material (endotoxin), and the production method using L cells is not preferable in terms of economics, such as the yield is very low (200㎍ / L) Can not do it.

Therefore, the inventors of the present invention, while researching to produce a large amount of midkine which is expected to be widely used as a growth factor that has neurostimulatory activity and regulates the growth and differentiation of cells, the gene of human midkine Was combined with the ubiquitin gene to successfully express the human midkine protein in yeast by genetic engineering methods.

Ubiquitin was discovered in 1975 and is found in eukaryotic cells as a whole and in the prokaryotic cells is reported to be absent (Goldstein, et al., Proc. Natl. Acad. Sci. 72, 1 (1975)). So far, the ubiquitin found in animal cells has been found to be the same, and there are various roles of ubiquitin, among which proteolysis is known to be important (Finley, et al., Trends in Biochem, Sci. 16, 343 (1985)). ).

In the yeast ubiquitin system, ubiquitin, consisting of 76 amino acids, is known to have a much faster intracellular cleavage process than other forms of ubiquitin and to cleave following arginine-glycine-glycine very efficiently (Ozkaynak, et al., Nature 312, 663-666 (1987). Also, Bachmair et al. Reported that foreign proteins bound to ubiquitin are cleaved very precisely after arginine-glycine-glycine by enzymes present in cells (Bachmair, et al., Science 234, 178-186 ( 1986)). Therefore, when the midkine gene is bound to the 3′-terminus of the ubiquitin gene and expressed in yeast, the ubiquitin gene induces the mass expression of the midkine protein and the midkine gene product is separated from the ubiquitin protein by an enzyme present in the cell. The desired human midkine protein can be obtained in large quantities.

Synthesizing a primer from the human midkine gene sequencing information to combine the midkine gene and ubiquitin gene as described above, amplify the human midkine gene in large quantities and clone it into an expression vector to fit the reading frame. By transfecting yeast with an expression vector and culturing it, a large amount of human midkine protein can be produced, contributing to the development of drugs related to nerve growth factor in the future.

Accordingly, an object of the present invention is to provide a method for expressing a human midkine protein by culturing an expression vector, a yeast transformed with the vector, and the transformed yeast containing the human midkine gene bound to the ubiquitin gene. In addition, it will further contribute to the development of neurostimulator-related drugs such as Alzheimer's and Parkinson's.

Hereinafter, the present invention will be described in detail.

Human midkine according to the present invention has an amino acid sequence as shown in FIG. In FIG. 1 amino acids are shown according to single letter notation.

The amino acid sequence of the human midkine of the present invention also includes an amino acid sequence substituted with an amino acid that does not affect the activity and function of the protein.

The DNA encoding human midkine of the present invention can be obtained directly from a cDNA library or synthesized chemically according to known methods. Any of several known methods can be used, such as, for example, phosphosamidite solid support methods such as Matteucci (J. Am. Chem. Soc. 103, 3185 (1981)). have.

In general, due to the degeneracy of the codons, a large number of codons corresponding to one amino acid exist, so that the DNA sequence encoding the amino acid sequence of the present invention may be partially different. The ubiquitin gene may be one that has been previously cloned or synthesized by chemical synthesis. The method for linking the ubiquitin gene and the midkine gene can be used by selecting and combining various known methods such as restriction enzymes, ligase, PCR, and DNA synthesis.

For example, first, ribonucleic acid (RNA) is extracted from the human fetal brain, and the oligonucleotide (RNA) is raised and cDNA is generated by reverse transcriptase using d (T) primer. On the other hand, polymerases corresponding to the 5 ′ and 3 ′ ends of the midkine gene encoding human midkine protein from the sequence information of human midkine (Kadomatsu, K. et al., BBRC 151, 1312 (1988)). Design and synthesize primers for polymerase chain reaction (PCR). These primers have a restriction enzyme recognition site at the 5 'end to allow binding to the ubiquitin gene, and the corresponding primer inserts the end codon and restriction enzyme recognition site to synthesize proteins from the starting codon of ubiquitin. Protein synthesis is completed at the codon inserted into the codon and facilitated cloning into the expression vector.

These primers are used to amplify the human midkine gene by a polymerase chain reaction using human fetal brain cDNA as a template, and a gene fragment obtained by cleaving the amplified gene with a restriction enzyme is used in an appropriate yeast expression vector, for example, the present applicant. The expression vector was prepared by substituting the HGH gene of pYLBC-A / G-UB-HGH (ATCC 74074, dated June 18, 1991), which was a previously applied yeast expression vector, and transformed the yeast with the recombinant vector. After that, the transformed yeast is cultured under conditions suitable for expression of the fusion gene. The production of the fusion protein can be commonly confirmed by SDS-polyacrylamide gel electrophoresis, and the resulting fusion protein can be separated and purified from cultured yeast cells or culture by a known method.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples merely illustrate the invention and do not limit the scope of the invention.

Experimental methods and procedures used in the following examples were carried out according to the following Reference Examples 1-6 unless otherwise specified.

Reference Example 1

DNA cleavage using restriction enzymes

Restriction enzyme and reaction buffer were added to a sterile 1.5 ml eppendorf tube to a reaction volume of 50 µl to 100 µl and reacted at 37 ° C. for 1 to 2 hours. After the reaction was completed, heat treatment was performed at 65 ° C. for 15 minutes to inactivate the restriction enzyme, and phenol extraction and ethanol precipitation were used for the restriction enzyme which was heat resistant. Restriction enzymes and reaction buffers used were purchased from New England Biolabs, MA, USA. The composition of the 10-fold reaction buffer is as follows.

Reference Example 2

[Phenol Extraction and Ethanol Precipitation]

Phenol extraction was used to deactivate the restriction enzyme or to extract hexane after the restriction enzyme reaction was completed. Phenol was used after saturation with 10 mM Tris-HCL (pH 8.0), 1 mM EDTA solution.

The sample and phenol are mixed at a ratio of 1: 1 (v / v), vigorously mixed, and then centrifuged (15,000 G, 5 minutes) to transfer the supernatant to a new tube. The same procedure was repeated three times, followed by extracting the supernatant with an equal volume of chloroform solution (chloroform to isobutanol ratio 24: 1 (/ v /)), adding 0.1 volume of 3M sodium acetate and 2.5 volume of 100% ethanol. do. This was allowed to stand at -70 ° C for 30 minutes or at -20 ° C for at least about 2 hours, followed by centrifugation (15,000xG, 20 minutes, 4 ° C) to precipitate hexane.

Reference Example 3

[Ligation]

T4 DNA ligase (T4 DNA ligase, New England Biolabs, Inc.) and 10-fold ligation buffer (0.5M Tris-HCl, pH7.8, 0.1M MgCl ₂ , 0.2M DTT, 10mM ATP, 0.5mg) as ligase / Ml BSA) was used to perform the ligation reaction. Usually, 10 units of ligase is used in a volume of 20 μl, and 100 units of ligase are used for ligation of DNA having blunt ends. At least 5 hours at 16 ° C. or 14 hours at 4 ° C. After the reaction was completed, the ligase was inactivated by heating at 65 ° C. for 15 minutes.

Reference Example 4

[E. coli transformation]

E. coli host cells (E. coli HB101, W3110 or JM105) were inoculated into 100 ml of liquid LB medium (1% bactotrypton, 0.5% bacterium yeast extract, 0.5% sodium chloride) and the optical density (OD) value at 650 mm was 0.25. Incubated at 37 ° C. until reaching 0.5. Cells were then separated by centrifugation (2,500 × G, 10 minutes) and then washed by adding 50 ml of 0.1 M MgCl ₂ . It was centrifuged again (2,500 × G, 10 minutes) and 50 ml 0.1M CaCl ₂ , 0.0.5M MgCl ₂ solution was added thereto and left to stand on ice for 30 minutes. This was again centrifuged (2,500 × G, 10 minutes) and uniformly dispersed in 5 ml of the same solution (0.1M CaCl ₂ , 0.05M MgCl ₂ ). All solutions and vessels used above were used after cooling at 0 ° C. 10 μl of the coupling reaction solution was added to 0.2 ml of the solution obtained above, and the mixture was left at 0 ° C. for 40 minutes and then heat-treated at 42 ° C. for 1 minute. This was added to 2.0 ml of liquid LB medium and incubated at 37 ° C. for 1 hour, and the culture solution was plated on solid LB medium containing 50 μg / ml ampicillin and then incubated at 37 ° C. overnight. M13 vector DNA was subjected to heat treatment after 100 μl of JM105 cells, 15 μl of 100 mM IPTG (isopropyl-β-thiogalactoside), 25 μl of 4% X-Gal (5-bromo-4-chloro-indolyl-β-galactoside) and Add 3 ml of double soft YT medium (1% NaCl, 1% yeast extract, 1.6% Bakto tryptone, 8.7% Bakto agar) preheated to 48 ° C, mix well, then double solid YT medium (1% NaCl, 1% yeast extract, 1.6% Bakto tryptone, 1.5% Bakto agar).

Reference Example 5

[Synthesis of oligomers]

The oligomers were synthesized by a DNA synthesizer (Applied Biosystems, Inc., model 380 B, USA) using an automated solid phase phosphoa midite chemistry and then modified polyacrylamide gel (2M urea, 12%). It was isolated by electrophoresis using acliamide, bis (29: 1 w / w) in 50 mM Tris, 50 mM boric acid, 1 mM EDTA-NA ₂ ). Next, it was attached to SEP-PAk (Waters Inc., USA), a C18 column, and purified using pure acetonitrile: water (50:50) as an eluent, and the concentration was determined by measuring absorbance at 260 mm.

Reference Example 6

[Polymerase Chain Reaction (PCR)]

Template DNA (10 ng to 100 ng), 10 μg of 10-fold Taq polymerase buffer solution (10 mM Tris-Cl pH8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.1% (w / v) gelatin), 10 μl of dNTP mixed solution (10 g each of dGTp, dATP, dTTP, and dCTP), 2 µg of primer, and 0.5 µl of AmpliTaq DNA polymerase (Perkin Elmer Cetus, USA) were added to distilled water to make 100 µl of the total volume. 50 μl of mineral oil was added to prevent evaporation of the solution. A temperature cycler (Perking Elmer Cetus, USA) was used, and the cycle program was repeated for 1 minute at 95 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C, and finally further reacted at 72 ° C for 10 minutes.

Example 1

[Amplification of Human Midkine Gene]

[Step 1]

RNA was extracted from the human fetal brain according to the method of Colozynski (P.) and Sacchi, N. (Anal. Biochem. 162, 156 (1987)). To a gram of human tissue, 10 ml of RNA extract (4M guanidinium thiocyanate; 25 mM sodium citrate, pH 7.0; 0.5% Sarcosyl; 0.1m 2-mercaptoethanol) was added to the cell homogenizer (Glass- Teflon Homogenizer, Thomas Scientific, USA), followed by 8 ml of 2M sodium acetate (pH 4.0), 8 ml of phenol (BRL, saturated with distilled water), 1.6 ml of chloroform-isoamyl alcohol (49: 1 , v / v) was added and mixed well, followed by centrifugation at 4 ° C. for 15 minutes at 12,000 × G. The aqueous solution of the upper layer was left for about 1 hour and then centrifuged at 12,000 × G and 4 ° C. for 20 minutes to obtain an RNA precipitate. The precipitate was washed with 75% ethanol and dried under vacuum for 10 minutes and then dissolved in 500 μl of TE buffer (10 mM Tris, pH 7.5; 1 mM EDTA).

Using the obtained human fetal brain RNA as a template for reverse transcriptase, cDNA was prepared using Stratagen's Zap-cDNA synthesis kit (Cat No. 200400), wherein the primer for reverse transcriptase was oligo d (T) primer ( 5'-GAGAGAGAGAGAGAGAGAGAAACTAGTCTC GAG (T) ₁₈ -3 ') was synthesized using a nucleic acid synthesizer (Aplied Biosystems Inc. USA 380B model). To synthesize cDNA single strands, 18 µl of the human fetal brain RNA solution obtained above was added, 2 µl of 0.1 M CH ₃ HgOH was added thereto, and left at room temperature for 10 minutes to release the secondary structure of RNA, followed by 2 µl of 1M 2-. Mercaptoethanol was added and reacted for 5 minutes. 5 μl reverse transcriptase reaction solution (500 mM Tris-CHl, pH 8.3; 750 mM KCl; 30 mM MgCl ₂ ; 10 mM DTT), 2.5 μl of dNTP (dATP, dGTP, dTTP and d CTP), 2 μl of oligo d ( T) primer (1.5µg / µl), 15µl of diethylpyro-carbnate (DEPC) -treated distilled water, 1.0µl of RNase inhibitor (RNase inhibitor, 1u / µl, Promega, USA) and then left at room temperature for 10 minutes RNA is a template and primers of reverse transcriptase, and then well butge 2.5㎕ ^- adding (18U / ㎕, superscript RNase H reverse transcriptase, BRL, USA) to 1 h at 37 ℃ was synthesized cDNA strand.

[Step 2]

The following primers were synthesized to clone the human midkine gene into the yeast expression vector.

Primer MKUB: 5´-AGACTCCGCGGTGGTAAAAAGAAAATAAGTGAA-3´

(Which has a restriction enzyme SacII recognition site corresponding to the 5 ′ end and comprises 20 bases after the signal sequence of human midkine).

Precursor MKSAL: 5´-AAAAAGTCGACTAGTCCTTTCCCTTCCCTTTCTT-3´

(It has a stop codon to stop detoxification and at the same time has a restriction enzyme SalI recognition site).

[Step 3]

Into the reaction tube A, 1 ng of the human fetal brain cDNA obtained in step 1 was used as a template, and 2 μg of the primer MKUB and 2 μg of the primer MKSAL were added, followed by 10 μl of 10-fold polymerization buffer, 10 μl of 2 mM dNTP (2 mM dGTP and 2 mM dCTP, respectively). , 2mM dATP, 2mM dTTP), 2.5 unit Taq polymerase and distilled water were added so that the total volume was 100 μl, followed by 1 minute at 95 ° C. (modification step), 40 seconds at 55 ° C. (annealing step), 2 at 72 ° C. The polymerase chain reaction (hereinafter, referred to as PCR) was performed 40 times under the conditions of the minute (polymerization step). The PCR product obtained above was separated from the 5% polyacrylamide gel and confirmed to have amplified about 370 base pairs of DAN, and was purified from the polyacrylamide gel under the same conditions (hereinafter, this DNA fragment was referred to as fragment MK).

[Step 4]

Nucleotide sequence identification of the amplified gene was performed according to the method of Sanger et al. (Snager, F.P.N.A.S. 74, 5463 (1977)). First, add 2 µl of 10-fold Klennow buffer solution and 10 units of Klenow fragment (New England Biolabs, USA) to 1 µg of the amplified gene, and add distilled water to make the total volume 20 µl. Both ends of the amplified genes were blunt ends by reacting for 30 minutes at and extracted with phenol / chloroform and dissolved in 20 µl of TE solution.

2 μg of plasmid M13 mp18 was completely cleaved with restriction enzyme EcoRV under the condition of NEB buffer 2, and separated with 0.7% agarose gel to isolate and purified about 7 Kb nucleic acid fragments. This nucleic acid is hereinafter referred to as fragment M13-RV. Into the ligation vessel, 100 ng of the smoothed end obtained above, 100 ng fragment M13-RV, 2 µl of 10-fold ligation solution, 10 units of T4 DNA ligase and distilled water were added so that the total volume was 2 µl. Next, the reaction was carried out at 16 ° C. for 12 hours. After the reaction, E. coli JM105 (ATCC 47016) was transformed, and 8 μl of 0.2M IPTG solution was added to the transformed cells, and 100 μl of pre-cultured helper cells (E. coli JM015) and 3 ml of 2XYT supernatant (soft agar, 16 g of Bakto tryptone per 1 l, 10 g of yeast extract, 5 g of NaCl, 5 g of Bactoagar) and 25 μl of 4% X-gal solution were mixed and minimum plate medium A (Min A plate, per 1 l) 10.5 g K ₂ HPO ₄ , 1 g (NH 4) ₂ SO ₄ , 0.5 g sodium citrate, 12 g bactoagar, 1 ml 20% MgSO ₄ , 0.5 ml 1% Vit B, 10 ml 50% glucose ) Was applied evenly on the) and incubated for 12 hours at 37 ℃ produced a transparent plaque (pick) with a toothpick, and incubated in 2 ml 2XYT liquid medium at 37 ℃, 5 hours and centrifuged. In the obtained supernatant, ¼ volume of PEG / NaCl (20% polyethyleneglycol, 2.5M sodium chloride) was added, and then M13 phage was collected to extract a single stranded nucleic acid, and it was confirmed to have a human midkine sequence.

Example 2

[Production of Yeast Expression Vector]

2 μg of plasmid pYLBC-A / G-UB-HGH (applied date: June 18, 1991, accession number: ATCC 74071, Korean Patent Application No. 89-20200) previously filed by the applicant with the restriction enzymes PstI and SalI Under the condition of NEB buffer 2, 2 µg of the same plasmid was completely cut under the condition of NEB buffer 4 with restriction enzymes PstI and subtracting enzyme SacII, and separated with 0.7% agarose gel to separate about 9.8Kb and 3.4Kb fragments, respectively. Purified. These fragments are hereinafter referred to as fragment PL2 and fragment PT2, respectively.

The fragment MK obtained in step 3 of Example 1 was completely cleaved with restriction enzyme SalI and restriction enzyme SacII under the conditions of buffer 3, and then extracted with phenol / chloroform and dissolved in 20 µl of TE solution. This was named fragment MK-T2 / L and ligation was carried out as follows.

In the conjugation reaction tube, 100 ng fragment MK-T2 / L and 100 ng fragment PL2, dir 100 ng fragment PT2, 2 μl of 10-fold conjugation solution, 10 units of T4 DNA ligase and a total volume of 20 μl Distilled water was added so as to react for 12 hours at 16 ° C. After the reaction, E. coli HB101 (ATCC 33694) was transformed to obtain pYLBC-A / G-UB-MK containing fragment UBMK (see FIG. 2).

Example 3

[Expression of Human Midkine Gene in Yeast]

Recombinant plasmids were extracted in a large amount by alkaline lysis method (Ika-Horowicz, D., et al., Nucleic Acid Res. 9, 2989 (1981)) for transformation of yeast, and the recombinant plasmid into yeast was used. Transformation was based on Beggs (Nature, 275, 104 (1978)) and Hinnen et al. (Proc. Natl. Acad. Sci. USA 75, 1929 (1978)).

12 ml of overnight cultured yeast (S. cerevisiae DC04, Yeast Genetic Stock Center, University of California, Berkeley, Calif., USA) was inoculated into 50 ml of fresh YEPD (2% peptone, 1% yeast extract, 2% glucose). After shaking culture at 30 ℃ and centrifuged for 5 minutes at 5000rpm cell precipitate was shaken in 200ml of water and again centrifuged under the same conditions. The cell precipitate was shaken again with 20 ml of 1M sorbitol solution and centrifuged at 5000 rpm for 5 minutes. Precipitated cells were dissolved in 5 ml of SP (1 M sorbitol, 50 mM potassium phosphate, pH7.5), 5 μl of 1 M DTT, and then 50 mg of zymolase (10 mg / zymolase, 50% glycerol / 50% SP). ㎖) and incubated for 30 minutes in a gentle shaking condition (150rpm) at 30 ℃, extracting the cell solution from time to time, treated with 1/10 volume of 0.15 SDS under spiro-plast under an optical microscope The degree of l formation was investigated, and about 90% or more of the yeast cells were cultured to be a spiro flask (usually about 30 minutes), followed by centrifugation (15,000 rpm, 3 minutes) to obtain spiroplasm, 5 ml of 1M. After washing with sorbitol and 5 ml of STC (1M sorbitol, 10 mM Tris-HCl, pH7.5) respectively, it was dissolved in the final 1 ml of STC and used for transformation.

10 μg of plasmid and 12.5 μl of 2 × STC (two-fold concentrate of STC) were mixed well, and 50 μl of the spiroplast was added thereto, followed by incubation for 10 minutes at room temperature. After adding 500 μl of PEG / TC solution (40% PEG-4000, 10 mM CaCl ₂ , 10 mM Tris HCl, pH 7.5), the solution was left to stand for 10 minutes, followed by centrifugation (Dynac Centrifuge (3 minutes, 1500 rpm)). The precipitate was obtained and dissolved in 200 μl of 1 M sorbitol, followed by 7 ml of regenerated agar (3 g of bacto agar per 100 ml, 50 ml of 2 M sorbitol, 0.07 g of amino acid deficient yeast nitrogen substrate, 0.1 g of amino acid lacking leucine). Mixture, 0.4 ml of 50% glucose, 45 ml of water) were applied on a plate and incubated at 30 ° C. for 3 to 5 days to obtain a yeast colony transformed with the recombinant plasmid. It was named Saccharomyces cerevisiae DC04-UB-MK, pYLBC-A / G-UB-MK and deposited with the ATCC on 8 December 1993 (Accession Number: ATCC 74259).

The transformed yeast strain was cultured lacking 3 ml of leucine (yeast nitrogen base without amino acids.Difco, USA) per 1 liter of culture medium, 0.25 g of leucine-deficient amino acid mixture. And 5% glucose), incubated at 30 ° C. for 24 hours, followed by centrifugation (15000 rpm, 3 minutes) to obtain yeast cell concentrates, and then suspended in YEP (2% peptone, 1% yeast extract) containing 2% glucose. After 24 hours and 48 hours of incubation, each culture solution was centrifuged and suspended in 400 µl of buffer solution (10 mM Tris-HCl, pH7.5, 1 mM PMSF (phenylmethylsulfonyl fluoride), 8M urea), and diameter 0.4. The glass beads of mm were added in the same volume and shaken vigorously to destroy the cells to obtain yeast chuchu rule. 10 μl of yeast extract was electrophoresed with 15% SDS-polyacrylamide gel according to the Laemmli, et al., Nature 227, 680 (1970), followed by staining with Coomassie brilliant blue R250. It was confirmed that human midkine protein was produced. According to the invention human midkine protein was produced at a level of 2 g / L, ie about 20% of the total protein (see Figure 3). In FIG. 3, the first row is yeast containing no plasmid, the second row is yeast containing plasmid pYLBC-A / G-UB-MK, and the sample is taken after 24 hours of incubation, and the third row is plasmid Samples taken after 48 hours of cultivation as yeast containing pYLBC-A / G-UB-MK are shown.

The biological activity of the human midkine protein produced according to the invention is identical to that of the existing Muramatsu method as described in patent application ________, entitled “Methods for Purifying Midcaine from Recombinant Yeast”. It was confirmed to be the same as that produced by

From the above results, it is possible to economically mass-produce midkine at higher yield than the conventional method reported in terms of the removal of pyrogenic substance and yield in terms of yield of recombinant yeast, thereby contributing to the development of neurostimulator-related medicine. It became possible.

Claims (4)

Expression vector pYLBC-A / G-UB / MK comprising a recombinant gene in which a human midkine gene is fused to a ubiquitin gene. Yeast transformed with the expression vector of claim 1. The yeast of Claim 2 which is Saccharomyces cerevisiae DC04-UM-MK, pYLBC-A / G-UB / MK. (Accession Number: ATCC 74259). A method for producing human midkine protein, comprising culturing the yeast of claim 2.