KR0135390B1 - Victors for expressing human pleiotrophin gene in yeast and - Google Patents

Abstract

The present invention relates to a yeast expression vector comprising a human fluorotrophin gene fused to a ubiquitin gene, a yeast cell transformed by the expression vector and a method for producing a human fluorotrophin protein using the yeast cell.

Description

Expression Vector of Human Pherotropin Gene and Method for Preparing Human Pherotropin Protein in Yeast

Figure 1 shows the nucleotide sequence of the human fluorotrophin gene and the amino acid sequence encoded therein,

Figure 2 shows the cloning process of the human fluorotrophin gene into the yeast expression vector,

Figure 3 shows the results of polyacrylamide gel electrophoresis of human fluorotropin expressed in yeast.

The present invention relates to the expression in the yeast of the human pleiotrophin gene.

More specifically, in the yeast expression vector comprising the human fluorotrophin gene fused to the ubiquitin gene, yeast cells transformed with the four-rok expression vector, and the method for producing the intercalotropin protein by culturing the yeast cells It is about.

Growth factors (cytokines or growth factors) play an important role in the regulation of cell growth and regulation of cell differentiation (Slack et al., Nature 326, 197-200 (1987); Rosa et al., Science 239, 783-785 (1988)). Murata et al., Proc. Natl, Acad. Sci. 85, 2434-2438 (1988); Thomas et al., Nature 346, 847-850 (1990); Mcmahon et al., Cell 62 ,. 1073-1085 (1990)), The fibroblasts (FGF), known as the family of one of the growth factors, are composed of several growth factors such as basic FGF, acidic FGF, int-2 and hst, and heparin binding growth factor because they have heparin binding activity. It is called.

Recently, a new heparin-binding growth factor family with structural differences from FGFs was discovered, and iotropin was first identified as an 18KD protein immunologically associated with acidic FGF, with heparin-binding activity in the bovine brain. Several laboratories have been reported under different names (for example, Heparin-Affinity Regulatory Protein (HARP), Heparin-Binding Growth Associated Molecule (MB-GAM), Heparin-Binding Neurotrophic Factor (HBNF), Heparin-Binding Growth Factor) (HBGF), Osteoblast-Specific Factor-1 (OSF-1), and the like.

Pliotropin is expressed during development in tissues derived from the brain and mesenchyme and is known as a polypeptide that induces growth and neurite triggers (Li, YS et al., Science 250, 1690-). 1694 (1990). This is a new field of research that has recently been studied as a new gene family along with midkine (MK) and retinoic acid-induced retinoic acid inducible heparin-binding protein (RI). These proteins are known to be involved in differentiation during the development of neuroectodderm, and gene expression in adults is found only in limited parts of the nervous system (Bohlen, P. and Kobes, I., Prog, Growth Factor Res. 3. 143-152 (1991) Subsequently, pleurophine was secreted as a growth factor from human breast cancer cells, and relatively high amounts were expressed in human melanoma cells (Wellstein, A.). , et al., J. Bio. Chem. 267,2582-2587 (1992)) This suggests that not only the development of iotropin but also the function of tumor growth, ie neuronal growth factor, can be suggested. (Lai, S. et al., Biochem. Biophys. Res. Commun. 187, 1113-1122 (1992).) Indeed, in 1993, Pyrotropin-expressing cells were expressed in nude mice after NIH 3T3 cells were expressed. Has been reported to confirm tumor formation. (Chauhan, AK, Proc. Natl. Acad Sci. Halde SA 90, 679 (1993)) The sequencing of the fluorotrophin cDNA revealed that the size of the mRNA of the fluorotrophin consists of 1024 bases and protein synthesis. The initiation codon encodes an 18.90 kilodalton polypeptide consisting of 168 amino acids by a 504 base size open reading frame (ORF) starting at and at the 178 base position.

The mature active fluorotrophin consists of 136 amino acid residues and is rich in basic amino acids with lysine clusters arranged at the amino and carboxyl ends (Merenmies et al., J. Biol. Chem. 265, 16721-). 16724 (1990). Although human polotropin proteins have been directly purified from tissues, their expression is limited to the brain and can only be purified from brain tissue extracts. Separation is also difficult and the amount of platelopin protein extracted is very small. In recent years, the human playotropin gene is cloned to induce the expression in E. coli, which also has a problem that it is not easy to remove the endotoxin derived from E. coli. Therefore, the inventors of the present invention ubiquitated the human playotropin gene during research to produce a large amount of human playotropin, which is expected to be widely used as a growth factor that has neurostimulatory activity and regulates cell growth and differentiation. Coupling with the quitin gene has been successful in the mass expression of human fluorotrophin protein in yeast by genetic engineering methods.

Ubiquitin, meanwhile, was discovered in 1975 and is found in eukaryotic cells as a whole and without its genes in primary cells (Goldstein, et al., Proc. Natl. Acad. Sci. 72, 11 (1975). The ubiquitins found in animal cells were found to be identical, and ubiquitin has several roles, among which proteolysis is known to be important (Finely, et al., Trands in Biochem. Sci. 16, 343 (1985)). In the yeast ubiquitin system, the 76-amino acid ubiquitin is known to have a much faster intracellular cleavage process than other forms of ubiquitin, and it is known to cleave following arginine-glycine-glycine very efficiently (Ozkaynak, et al., Nature). 312, 663-666 (1987)), and Bachmair et al. Also found that foreign proteins bound to ubiquitin by enzymes present in cells are known as arginine-glycine-. It has been reported that it is cleaved very accurately after lysine (Bachmair, et al., Science 234, 178-186 (1986)), therefore, when the desired foreign protein gene is combined with the ubiquitin gene and expressed in yeast, The desired foreign protein is induced in large quantities, and the desired protein is separated from the ubiquitin by the ubiquitin cleavage enzyme present in the cell, so that only the protein can be obtained in large quantities.

It is therefore an object of the present invention to provide an expression vector linking the human fluorotrophin gene to the 3 'end of the ubiquitin gene, and further promoting the future nerve by mass expression of the human fluorotrophin protein in yeast transformed with the vector. It contributes to the development of medicines related to factors.

Hereinafter, the present invention will be described in detail.

The fusion gene linked to the ubiquitin gene and the human playotropin gene for the production of the expression vector of the present invention is a restriction enzyme cleavage method, a linkage by DNA ligase, an appropriate primer, by using a cloned gene or cloned gene. PCR can be prepared using a variety of methods known in the art, such as chemical synthesis of DNA, and the fusion gene is prepared according to the type of yeast host cell to be used according to the type of expression of the appropriate vector after selecting the vector One known genetic engineering method is used to insert the vector into a yeast expression vector of the human playotropin gene.

For example, to clone the human fluorotrophin gene, ribonucleic acid (RNA) was extracted from dead human fetal brain cells to synthesize whole cDNA using this RNA, random primers, and reverse transcriptase. The primers designed from the nucleotide sequence of the otropin gene are used to amplify the human playotropin gene in human fetal brain cells. The amplified human fluorotrophin gene may be prepared by cleavage using an appropriate restriction enzyme and then inserting it after the ubiquitin gene of a vector prepared to include the ubiquitin gene.

The expression vector thus prepared is transformed into a suitable yeast host cell such as Saccharomyces cerevisiae, and the yeast transformant containing the expression vector is allowed to express the playotropin gene. Cultured under the above conditions to produce human fluorotrophin protein.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are only intended to give specific details and do not limit the scope of the present 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

The sterilized 1.5ml was added to the reaction volume of 50 μl to 100 μl of the restriction enzyme and the reaction buffer solution in the Epondorf tube and reacted at 37 ° C. for 1 to 2 hours. After the reaction was completed, 15 minutes heat treatment at 65 ℃ to inactivate the restriction enzyme, phenol extraction and ethanol precipitation method was used for the heat-resistant restriction enzyme.

Restriction enzymes and reaction buffers used were purchased from New England Biolab, MA, USA. The composition of the 10-fold reaction buffer is as follows.

10-fold NEB buffer 1: 100 mM bis tris propane-HCl, 100 mM MgCl 2,

10 mM dithiothreitol (DTT), pH 7.0

pH 7.0

10-fold NEB buffer 2: 100 mM Tris-HCl, 100 mM Mgcl2, 500 mM NaCl,

10 mM DTT, pH 7.0

10-fold NEB buffer 3: 100 mM Tris-HCl, 100 mM MgCl 2, 1000 mM NaCl,

10 mM DTT, pH 7.0

10x NEB buffer 4: 200 mM-acetate, 100 mM magnesium

Acetate, 500 mM Potassium Acetate, 10 mM

DTT, pH 7.0

Reference Example 2

Phenolic Extraction and Ethanol Precipitation

Phenol extraction was used to deactivate the restriction enzyme or to extract the nucleic acid 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), shaken vigorously, mixed, and centrifuged (15,000 × G, 5) to transfer the supernatant to a new tube. The procedure was repeated three times, followed by extracting the conflict solution with the same volume of chloroform solution (chloroform to isobutanol ratio 24: 1 (v / v)), and adding 0.1 volume of 3M sodium acetate and 2.5 volume of 100% ethanol. This was allowed to stand at -70 ° C for 30 minutes or at -20 ° C for at least about 2 hours, followed by centrifugation (15,000 x G, 20 minutes, 4 ° C) to precipitate the nucleic acid.

Reference Example 3

Ligation

T4 DNA ligase (T4 DNA ligase, New England Biolabls, Inc.) and 10-fold ligation buffer (0.5M Tris-HCl, pH 7.8, 0.1M MaCl2, 0.2M DTT, 10mM ATP, 0.5mg / ml as ligase) BSA) was used to perform the ligation reaction. Normally, 10 units of ligase is used at a volume of 20 μl, and 100 units of ligase are used for ligation of DNA having blunt ends, and reaction is performed for 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, 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 nm was 0.25. Incubate at 37 ° C. until reaching 0.5. Cells are 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 x G, 10 minutes), and 50 ml 0.1M CaCl2, 0.05M MgCl2 solution was added thereto and allowed 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). 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 a solid LB medium containing 50 μg / ml ampicillin and then incubated overnight at 37 ° C. M13 vector DNA was heat treated and then 100 μl of JM105 cells, 15 μl of 100 mM IPTG, 25 μl of 4% X-Gal and 3 ml of 2x soft solid YT medium (1% NaCl, 1% yeast extract, 1.6) preheated to 48 ° C. % Bakto tryptone, 8.7% Bakto agar) was added and mixed well, and then plated on a 2-fold solid YT medium (1% NaCl, 1% enzyme extract, 1.6% Bakto tryptone, 1.5% Bactoagar).

Reference Example 5

Synthesis of Oligomers

The oligomer was synthesized with a DNA synthesizer (Applied Bioystems, Inc., mode 380 B, HaldeSA) using an automated solid phases phosphoamidite chemistry and then modified polyacrylamide gel (2M urea, 12% acrylamide, bis (29: 1 w / w) in 50 mM Tris, 50 mM boric acid, 1 mM EDTA-Na2) was separated by electrophoresis. Next, it was attached to SEP-PAK (Waters Inc., Halde S. A), which is a C18 column, and then purified by using acetonitrile: water (50:50) as an eluent and measuring the absorbance at 260 mm. Decided.

Reference Example 6

Polymerase Chain Reaction (PCR)

Template DNA (10 ng to 100 ng), 10 μl of 10-fold Taqwndgkqgyth buffer (10 mM Tris-Cl pH 8.3, 500 mM KC1, 15 mM MgCl2, 0.1% (w / v) gelatin), 10 μl of dNTP mixture (dGTP, dATP, dTTP) , dCTP each 10mM), 2μg each primer (primer), 0.5μl Amplitaq DNA polymerase (Perkin Elmer Cetus, USA) was added to distilled water to a total volume of 100μl. To this was added 50 μl of mineral oil to prevent evaporation of the solution. The temperature cycler (Perkin Elmer Cetus, U.S.A) 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

RNA and cDNA Synthesis of Human Fetal Brain Cells

2 g of dead human fetal brain cells were mixed with 20 ml RNAzol B (chomczynski, et. Al., Anal Biochem. 162. 150-159 (1987)), left at 4 ° C. for 10 minutes, and treated with vortex for 1 minute. Add 2 ml of chloroform: isoamyl alcohol (29: 1v / v), mix, and centrifuge at 4 ° C for 15 minutes (using Beckman JA-13rotor) to collect the supernatant and add isopropyl alcohol in the same volume. The mixture was left at 4 ° C. for 15 minutes, centrifuged at 4 ° C. 12000 rpm for 15 minutes to precipitate RNA, and then dissolved in DEPC (diethylpyrocarbonate) -treated distilled water. 10 μg of the RNA thus obtained was treated with 5 drops at 65 ° C. and immediately placed on ice. 20 μl of 5 mM dNTP (5 mM each of dATP, dGTP, dCTP and dTTP) and 40 μl of 5 × RT Buffer (5X reverse transcriptase bugger; 0.25M Tris -HCl, pH 8.3, 0.5M KCl, 50mM KC1, 50mM MgCl2) After adding 10μ 0.5mg / ml random primer and 80μl H2O, 10μ1 RNasinPromega Biortech USA 1U / μl) and 20μ M-MLV reverse transcriptase The mixture was stirred well and reacted at 37 ° C. for 60 minutes. Subsequently, 5 μl of 0.5M EDTA (pH 8.0) and 200 μl of phenol were added and stirred well, followed by centrifugation at room temperature for 10 minutes to obtain a supernatant. 20 μl of 3M sodium acetate and 1 ml of 95% ethanol were added thereto. Participated in the war.

Example 2

Cloning of the Playotropin Gene

2-1) Design of primer

The following primers were made to clone the playotropin gene. The primer UBHPTNT2 (5'-GAGACTCCGCGGTGGTGGGAAGAAAGAGAACC-3 ') is a 5'-side primer starting from the glycine codon (94th base from ATG) corresponding to the first amino acid of the genotyped polotropin gene. Containing the required SacII restriction enzyme recognition site, the primer HPTNLTI (5'-ACTCGAGTCGACTATTAATCCAGCATCTTCTCCTG-3 ') has the recognition site of restriction enzyme SaII for cloning, including a termination codon as the 3'-side primer of iotropin. have.

2-2) Amplification of Playotropin Gene

1 μg of the primers UBHPTN2 and HPTNL1 designed in Example 2-1) were added to the reaction tube, and then 50 ng of the cDNA made in Example 1, 10 μl of 10 times polymerization buffer solution, 10 μl of 10 mM dNTP (dATP, dGTP, dTTP, dCTP respectively 10mM) and 2.5 unit Taq polymerase was added and distilled water was added to make a final volume of 100μl and PCR reaction was carried out 25 times as shown in 6. This PCR product was separated with 7% polyacrylamide gel, and purified by DNA extraction of about 410 base pairs, dissolved in 20 μl TE solution, and completely cleaved with restriction enzyme SacII under 4 conditions of NEB buffer solution and SalI under 3 conditions of NEB buffer solution After cleavage, the residue was purified with 7% polyacrylamide gel and cleaved into 20 μl of TE, followed by purification with 7% polyacrylamide gel and dissolved in 20 μl of TE. This fragment is hereinafter referred to as HPTN. Sequence identification of the amplified gene was performed according to the method of Sanger et al. (Sanger, F. et al., PNAS 74, 5463 (1977) (Fig. 1).

Plasmid pYLBC0A / G-UB-HGH (see patent application 89-20200; ATCC 74071, dated June 18, 1991) was intactly cleaved under the conditions of NEB buffer 3 with restriction enzymes Pst I and SalI. In addition, 2 μg of the same plasmid was cut completely with the restriction enzymes PstI and SacII under the conditions of NEB buffer 4 to separate and purify 9.8 kb and 3.4 kb fragments in 0.7% agarose gel, respectively. These fragments are hereinafter referred to as fragments PL2 and PT2, respectively.

2-3) Preparation of Expression Vectors

100ngTlr of fragment HPTN and 100g of fragment PL2 PT2 obtained in Example 2-2) were added, 2μl of 10-fold conjugation buffer solution, 10-unit T4 DNA ligase, and distilled water were added so that the total volume was 20μl. It reacted at 12 degreeC. After the reaction, E. coli HB101 (ATCC 33694) was transformed to obtain pYLBC-A / G-UB-HPTN containing fragment HPTN (Figure 2).

Example 3

Playotropin Gene Expression in Yeast

Recombinant plasmid pYLBC-A / G-HPTN was extracted in a large amount by an alkaline elution method (Ish-Horowicz, D., et. Al., Nucl.Acid.Res. 9, 2989 (1981) and the yeast (Saccharomycese cerevisiae DC04) Yeast genetic Stock Center, University of California, Berkeley, Calif., USA, the method of literature (Baggs, Nature 275, 104 (1978); Hinnen, Proc. Natl. Acad. Sci. USA 75, 1929 (1978)). Thus transformed as follows.

12 ml of yeast cultured overnight was inoculated into 50 ml of fresh YEPD (2% peptone, 1% yeast extract, 2% glucose) and shake-cultured at 30 ° C. until the OD (optical density) value was 0.8 to 1 at 650 nm. The cell precipitate obtained by centrifuging the culture solution at 5000 rpm for 5 minutes was shaken in 20 ml of water, and then ndjstla was separated again. The cell precipitate was shaken again with 20 ml of 1M sorbitol solution and recentrifuged. Precipitated cells were added 5ml of SP (1M sorbitol, 50mM / m in 50% glycerol / 50% SP) with 5μl of 1M DTT and incubated for 30 minutes in a gentle shaking condition (150rpm) at 30 ℃, From time to time, the culture solution was extracted and treated with 1/10 volume of 0.1% SDS to investigate the degree of spheroplast formation under optical microscope observation. (1500 rpm, 3 minutes) to obtain a spiro flask and then washed in 5ml 1M sorbitol and 5ml STC (1M sorbitol, 10mM Tris HCl pH 7.5), respectively, dissolved in the final 1ml STC and used for transformation. Mix 12.5μl 10μg plasmid and 12.5μl 2 × STC (double concentration of STC), add 50μl of spheroplasmol prepared above, incubate for 10 minutes at room temperature, and 500μl PEG / TC solution (40% PEG 4000, 10 mM CaCl 2, 10 mM Tris HCl pH 7.5) was added and then left to stand for 10 minutes. The cells were precipitated by centrifugation (3 minutes at 1500 rpm with a Dynamic Centrifuge) and dissolved in 1M sorbitol. 7 ml of regeneration agar (3 g of bactoa per 100 ml, 50 ml of 2M sorbitol, 0.07 g of yeast nitrogen substrate without amino acid, 0.1 g of amino acid mixture yeast nitrogen substrate lacking leucine, 0.1 g of leucine lacking) Amino acid mixture, 0.4 ml of 505 glucose, 45 ml of distilled water, and the like) were incubated at 30 ° C. for 3-5 days to obtain a yeast clone transformed with the recombinant plasmid. Yeast Saccharomyces cerevisiae DC04, transformed with plasmid pYLBC-AG-HPTN, was deposited on February 3, 1994, under the ATCC 74267 to the American Type Culture Collection (ATCC).

0.25 g of a culture solution lacking leucine of 3 ml of transformed yeast strain (Yeast nitrogen base without amino acid, Difco, USA) 6.7 and leucine deficient amino acid mixture 0.25 g and 5% glucose), incubated for 24 hours at 30 ℃ centrifugation (3 minutes at 1500rpm) and suspended in 400μldhkscnddydor (10mM Tris HCl, pH 7.5, 1mM PMSF (phenylmethylsulfonylfluoride), 8M urea) and 0.4mm in diameter glass The beads were added in the same volume and shaken vigorously to destroy the cells to obtain a yeast extract. 10 μl of yeast extract was electrophoresed on 15% polyacrylamide gel according to the method of Laemmli, Nature 227, 680 (1970) and stained with Coomassie brilliant blue R250 to give human fluorotropin protein. This mass was confirmed to be produced (FIG. 3).

In FIG. 3, the first row is a yeast containing plasmid pYLBC-A / G-UB-HPTN and is a sample taken after 24 hours, and the second row is a yeast containing plasmid pYLBC-A / G-UB-HPTN. The sample was taken again after 48 hours, and the third column represents yeast containing no plasmid.

From the above results, it can be seen that human fluorotrophin is produced in yeast in an amount of 30% or more of the total yeast protein.

Claims (4)

Yeast expression vector comprising a human playotropin gene linked to the ubiquitin gene. The expression vector according to claim 1, which is pYLBC-A / G-UB-HPTN. 4. The yeast Saccharomyces cerevisiae DC 04 (ATCC 74267) according to claim 3, transformed with pYLBC-A / G-UB-HPTN. Method for producing a human fluorotrophin protein, comprising culturing the yeast of claim 3 or 4.