KR920001745B1 - Expression vector for human growth hormone gene - Google Patents

Abstract

A process for preparing human growth hormone exprssion vector (PYLVC- A/G-UB-HGH) comprises (a) inserting primer 1 and primer 2 into yeast CDNA to synthesize ubiquitin gene (I) having restriction sites SacII and BstBI, (b) synthesizing promotor-gene-ubiquitin gne (II) having SacII, BamHI site from (I), HGH expression vector (PYLBC-A/G-HGH), primer 2 and primer 3, and (c) recloning (II), terminator gene-HGH gene having BamHI, XhoI site made from the above (PYLBC-A/G-HGH), and a linker having XhoI, SacII site into yeast expression carrier.

Description

Human growth hormone gene expression vector [pYLBC-A / G-UB-HGH] and its preparation method

Figure 1 shows the nucleotide sequence and amino acid sequence of human growth hormone linked to ubiquitin in the expression vector according to the present invention,

Figure 2 shows the position of the primer on the gene according to the present invention,

Figure 3 illustrates the cloning process into the yeast expression carrier according to the present invention,

4 shows the results of electrophoresis to confirm the expression of the human growth hormone gene by fermenting the yeast transformed with the human growth hormone expression vector [pYLBC-A / G-UB-HGH] of the present invention.

The present invention relates to a human growth hormone expression vector [pYLBC-A / G-UB-HGH] and a method for preparing the same, and more specifically, to a human growth hormone in a human body using a yeast as an expression agent according to genetic engineering methods. It relates to a human growth hormone expression vector [pYLBC-A / G-UB-HGH] which can be usefully used for mass production of the same human growth hormone and a method for producing such a vector.

Human growth hormone is a peptide hormone with a molecular weight of approximately 21,500 daltons consisting of 191 amino acid groups and has been mainly used for the treatment of dwarfism (Robin, MS, J. Clin. Endocr. 18, 901 (1958). ))

Conventionally, human growth hormone has been extracted and used mainly from the pituitary gland of humans who died from traffic accidents or diseases, but the amount is so limited that it has not been supplied to all dwarf patients and the price is high, and there are many difficulties in use.

In addition, four children who received human growth hormone extracted from the pituitary gland of a recently deceased human were infected with an unidentified virus and died of an accident. The FDA banned the use of human growth hormone extracted and purified from the dead pituitary gland as described above (Science 234, 22 (1986)).

On the other hand, as a method for manufacturing human growth hormone, chemical synthesis, tissue culture, or other microbial culturing methods by gene combinations are known, but these methods also have low yields, and the price has been increased due to unreasonable manufacturing processes. When manufacturing by the gene combination method of the N-terminal had a problem such as a separate process for removing extra methionine generated in the gene expression process.

Therefore, the present inventors have made efforts to solve the above problems, mass production of methionine-free human growth hormone by connecting the ubiquitin gene to human growth hormone gene using the characteristics of ubiquitin in yeast according to genetic engineering method It has been found that the present invention can be usefully used to complete the present invention.

That is, an object of the present invention is to provide a human growth hormone expression vector and a method for producing the same, which can be used for mass production of the same human growth hormone as human growth hormone in the human body.

Hereinafter, the present invention will be described in detail.

The present invention synthesizes the ubiquitin gene containing 30 effector nucleotide sequences having restriction enzymes SacII and BstB1 by adding primer 1 and primer 2 to the yeast chromosome gene, and the gene and human growth hormone expression vector [pYLBC-A / G-UB-HGH], and primers 2 and 3 synthesized the effector-ubiquitin gene having the Sac II, BamH I site, and then in this gene and the human growth hormone expression vector [pYLBC-A / G-UB-HGH] Preparation of Human Growth Hormone Expression Vector [pYLBC-A / G-UB-HGH] by Cloning Cloned End Gene-Human Growth Hormone Gene with BamHI, XhoI Site and Link Linker with XhoI, SacII Site in Yeast Expression Carrier The method is characterized by the present invention, and the present invention also features a human growth hormone expression vector [pYLBC-A / G-UB-HGH] prepared according to the method.

Referring to the present invention in more detail as follows.

The present invention is to connect the ubiquitin gene (UB) to the human growth hormone gene (HGH) so that when the human growth hormone gene is expressed in the yeast host can be obtained in the form of the N-terminal methionine is removed.

Such ubiquitin genes of the present invention are derived from yeast, in particular Saccharomyces cerevisiae AB110, and the human growth hormone gene has been deposited with the KPAC-10669 with the Korean spawn association in connection with patent application No. 88-18192, which is the applicant's prior application. It is derived from the human growth hormone expression vector [pYLBC-A / G-UB-HGH] of Saccharomyces cerevisiae [pYLBC-A / G-UB-HGH].

Hereinafter, the present invention will be described step by step according to the manufacturing method.

First, yeast (Saccharomyces cerevisiae AB110) was incubated in YEPD (1% yeast extract, 2% peptone, 2% glucose) medium and thus cultured from yeast by Struhl et al. (Nature, 305, 391 (1983)). Extract the entire nucleic acid.

Next, a total of 53-mers (5 ') were formed by concatenating 30 3′-terminal sequences of the ADH / GAP hybrid agonist (A / G) with 23 5′-terminal sequences of the ubiquitin gene (UB). Primer 1 of -GTTTCGAATAAACACACATAAACAAACACCATGCAAATTTTCGTCAAAACTCT-3 ') and Primer 2 of 28-mer (3'-GTGAACCACAACTCTGAGGCGCCACCA-5') complementary to the ubiquitin 3'-end upper DNA are prepared. The primers 1 and 2 were added to the whole yeast nucleic acid, followed by polymerase chain reaction (Polymerase chain Reaction: Saiki et al., Science 230, 1350 (1985)), which had 30 gene sequences and restriction enzymes SacII and BstBⅠ. A ubiquitin gene having a site (Engin Ozkaynak et al., Nature 312, 663 (1984)) is synthesized.

The gene and the human growth hormone expression vector [pYLBC-A / G-UB-HGH] obtained in this manner are located above the working genes of the primer 2 and the human growth hormone expression vector [pYLBC-A / G-UB-HGH]. Add primer 3 of 24-mer (5′-GCCACCATACCCACGCCAAACAA-3 ′) identical to the top strand sequence, and polymerase chain reaction again, containing the A / G agonist and the ubiquitin gene and having the SacII and BamHI sites. (SacII-BamHI section) is obtained.

On the other hand, the human growth hormone expression vector [pYLBC-A / G-UB-HGH] was digested with restriction enzymes Xho I and HamH I to contain human growth hormone genes and terminator genes and had Xho I and HamH I sites (Xho I-HamH I fragments). Get

Next, to connect the SacII-BamHI fragment and the XhoI-HamHI fragment obtained above, the upper strand 24-mer (5) is designed to be complementary to the SacII site of ubiquitin and the other to the XhoI site in the human growth hormone gene. A XhO-SacII linker consisting of ′ -GGTGGTTTCCCAACTATTCCATC-3 ′) and a lower strand 31-mer (3′-CGCCACCAAGGGTTGATAAGGTGAGAGCTG-5 ′) was prepared, and the SaCII-BamHI fragment and XhoI-HamHi segment with the XhoI-SacII linker. Under the addition of T ₄ DNA ligase, it is cleaved with BamHI and recloned into a yeast expression carrier [pYLBC] treated with BAP (Bacterial Alkaline Phosphatase).

As a result, the human growth hormone expression vector [pYLBC-A / G-UB-HGH] of the present invention, in which the human growth hormone gene and the ubiquitin gene are linked and containing A / G as a working gene, is obtained.

The present invention will be described in more detail based on the following examples.

Example 1

[Extraction of Whole Yeast Nucleic Acids]

After centrifugation of yeast (Sacharomycescerevisiae AB110) cultured in 50 ml of YEPD (1% yeast extract, 2% peptone, 2% glucose) for 24 hours, the cell precipitate was dissolved in 10 ml of 1M sorbitol. Then centrifuge again and add 5 ml of 1 M sorbitol, 50 mM Potassium phosphate (pH7.5) buffer and 5 μl of 1 M dithiothreitol (DTT, Sigma Chemical Co., USA) to the extracted precipitate. It was stirred. 1 mg of Zymolase (Zymolase, Miles Co., U.S.A) was added thereto, followed by reaction at 30 ° C. for 30 minutes.

The reaction mixture was centrifuged at 5,000 rpm (Beckman J6 Centrifuge, rotor JS4.2) for 5 minutes, and the cell precipitate was dissolved in 5 ml of TE (10 mM Tris HCl (pH 7.5), 1 mM EDTA) buffer solution. 0.5 ml 0.5 M EDTA and 0.25 ml 10% SDS were added. After reacting at 65 ° C. for 30 minutes, 1 ml of 5M potassium acetate was added thereto and reacted in ice for 1 hour.

It was then centrifuged for 25 minutes at 15,000 rpm (Beckman J21 centriguge, rotor JS13), the supernatant was separated and 15 ml of 95% ethanol was added thereto. After centrifugation at 15,000 rpm for 15 minutes, the resulting supernatant was discarded and the precipitate was vacuum dried.

This dried precipitate was dissolved in 5 ml of TE buffer, and then 20 μl of RNase (I (10 mg / ml, New England Lab., U.S.A.) was added and reacted at 37 ° C. for 30 minutes.

Then, extracted three times with phenol: chloroform: isoamyl alcohol (25: 24: 1V / V / V), the salt was added to the supernatant so that the final concentration was 0.3M, and the same volume of 95% ethanol was added. Next, the mixture was left at minus 20 ° C for at least 2 hours, and then centrifuged at 10,000 rpm for 30 minutes. In this way, the whole yeast nucleic acid was obtained. It was dissolved in 0.5ml TE buffer and used for the next experiment.

Example 2

[Amplification of Ubiquitin Gene by Polymerase Chain Reaction and Connection with Promoter]

The 5'-terminal and 3'-terminal sequences of all known ubiquitin genes were used to make primers for polymerase chain reaction.

As shown in FIG. 2, primer 1 is a total of 53-mers (5'-GTTTCGAATAAACACATAAACAAACACCATGCAAATTTTCGTCAAAACTCT-3 ') which connects 30 3'-terminal sequences of the A / G promoter with 23 5'-terminal sequences of ubiquitin. Synthesis was performed using a nucleic acid synthesizer (Applied Biosystems Inc. Moel 380B, USA). In addition, primer 2 was made of 28-mer (3′-GTGAACCACAACTCTGAGGCGCCACCA-5 ′), which is complementary to the ubiquitin 3′-top DNA strand, and was synthesized by altering the 219-222th nucleotide sequence for linkage with the human growth hormone gene. There was no change in the amino acid to generate the Sca II site.

The product was taken at 260 nm to have an O.D. value of 1, dried, dissolved in 200 μl of distilled water and used for the next reaction.

10 μl of 10 × Taq polymerase buffer solution (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl ₂ , 0.1% (W / V) gelatin) and 10 μl in 1 μl of yeast nucleic acid obtained in Example 1 DNTP ′S mixed solution (dGTP, dATP, dTTP, dCTP is 1.25 mM each), 10 μl of primer 1, 5 μl of primer 2, 63.5 μl of distilled water and 0.5 μl of Ampli Tap DNA polymerase (5 Unit / μl, Perkin-Elmer-Cetus, USA) was added and mixed well. At this time, 50 μl of mineral oil was added to the reaction solution to prevent evaporation of the solution. A temperature cycler (Tempcycler, Perkin Elmer-Cetus, USA) was used to perform the amplification reaction. The temperature cycle program was 30 seconds at 94 ° C; 30 sec at 50 ° C .: 25 cycles of 1 min at 72 ° C. were repeated and finally further reacted at 72 ° C. for 10 min.

The nucleic acid of the amplified ubiquitin was separated and purified by electrophoresis using 1.5% agarose gel, and then the following polymerase chain reaction was used. Polymerase chain reaction was again performed to link the amplified ubiquitin gene with the A / G hybrid effector.

Primer 3 of a 24-mer (5'-GCCACCATACCCACGCCGAAACAA-3 ') identical to the top strand sequence above the gene in the human growth hormone expression vector [pYLBC-A / G-UB-HGH] was synthesized.

Then, 10 μl of 10 × Taq polymerase buffer solution was added to 1 μl (10 pg / μl) of human growth hormone expression vector [pYLBC-A / G-UB-HGH] and 1 μl of purely isolated ubiquitin gene. dNTP ′S mixed solution, 10 μl of primer 2, 5 μl of primer 3 and 67.5 μl of distilled water were added, and 0.5 μl (5 Unit / μl) of Ampli Taq DNA polymerase was added to mix well.

As a result of the amplification reaction using the same temperature cycling program, the linked A / G working gene-ubiquitin nucleic acid was obtained.

The newly synthesized nucleic acid fragments were further purified by 1.5% agarose gel electrophoresis as described above, and then cut into BamH I and Sac II, which were used in the next conjugation reaction.

Example 3

Cloning into Yeast Expression Carriers

The human growth hormone expression vector [pYLBC-A / G-UB-HGH] was digested with restriction enzymes Xho I and Sal I, and the human growth hormone gene-GAP end gene fragment with 21 base pairs removed at the 5'-end was described as described above. Purification by Yeongdong

SacII-XhOI linkers of upper strand 24-mers (5'-GGTGGTTTCCCAACTATTCCACTC-3 ') and lower strand 31-mers (3'-CGCCACCAAAGGGTTGATAAGGTGAGAGCTG-5') designed to complement one of the SacII sites in ubiquitin were synthesized.

Yeast carrier pYLBC (100 ng, ATCC 37115, Cat.No.N338 from Amersham, USA or Yeas-Genetic Stock Center, University California, Berkeley, Calif., USA) cut with BamHI and the A / G working gene obtained in Example 2- BamHⅠ-SacⅡ nucleic acid fragments of ubiquitin gene (50ng), SacⅡ-XhoⅠ linker (10ng), and human growth hormone - for the end gene XhoⅠ-BamHⅠ nucleic acid fragment (50ng) T ₄ DNA referred to this rise (T ₄ DNA ligase, Biolab, USA), and then transformed with E. coli HB101 (ATCC 33698), and selected from an empicillin plate. The ubiquitin-human growth hormone gene was extracted from the recombinant colonies by alkaline digestion (Alkaliiysis, Birnboim and Doly, Nucleic Acids Res., 7, 1513 (1979)), and then plasmids were digested with restriction enzymes SacII and SalI to 580 base pairs. It was confirmed that the nucleic acid fragments were separated. The ubiquitin-human growth hormone nucleic acid fragment obtained by cleaving this recombinant plasmid [pYLBC-A / G-UB-HGH] with BstBI and SalI was cloned into M13 to obtain Messing et al. (Meth. Enzymol. Enzymol. 101, 20, 1983). The base sequence was confirmed by the method. The nucleotide sequence is shown in FIG.

As described above, the process of cloning into the yeast expression carrier is summarized in FIG.

The identified recombinant plasmids were extracted in a large amount by alkaline digestion and used for transformation into yeast. To transform the recombinant plasmid into yeast was performed according to the method of Beggs (Nature, 275, 104 (1978)) and Hinnen et al. (PNAS USA 75, 1929 (1978)). 1 ml of S. cerevisiae AB110 cultured overnight was inoculated into 50 ml of fresh YEPD medium and shaken at 30 ° C. until the OD ₆₅₀ value was 0.8 to 1.

After centrifuging the water at 5000 rpm for 5 minutes, the cell precipitate was shaken in 20 ml of water and centrifuged again. The cell precipitate was shaken again with 20 ml of 1M sorbitol solution and centrifuged again. Precipitated cells were well dissolved in 5 ml of SP (1 M sorbitol, 50 mM Potassium phosphate, pH 7.5) and 5 μl of 1 M DTT, followed by 50 μl of Zymolase (containing 10 mg / ml in 50% glycol / 50% SP). Put and incubated for 30 minutes under gentle shaking conditions (150rpm) at 30 ℃. At this time, the cell solution was extracted from time to time, treated with 1/10 volume of 0.1% SDS, and cultured until 90% or more of yeast cells became spiroflasks by examining the degree of spiroplast formation under an optical microscope ( Typically 30 minutes).

Next, spiroplast was obtained by centrifugation at 1500 rpm for 3 minutes with a Dynac centrifuge, followed by washing with 5 ml of 1 M sorbitol and 5 ml STC (1 M sorbitol, 10 mM CaCl ₂ , 10 mM Tris HCl, pH 7.5), respectively, and the final 5 ml STC. After lysis, it was used for transformation.

After mixing 12.5 μl of 5-10 μg plasmid nucleic acid and 12.5 μl of 2 × STC (two-fold concentrate of STC), 50 μl of the spiroplast prepared above was added and incubated at room temperature for 10 minutes.

500 μl of 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.

Spiroplast precipitate was obtained by centrifugation at 1500 rpm for 3 minutes with a Dynac centrifuge, which was well dissolved in 200 μl of 1M sorbitol.

7 ml of Regeneration Agar (3 g of Bakto-Agar per 100 ml, 50 ml of 2M sorbitol and 0.67 g of Yeast Nitrogen Base (W / Oamino acid), 0.1 g of Leu ^- feed, 0.4 ml of 50% glucose and containing 45ml of water) and the materials were mixed well, Leu ^- sorbitol plates + (182g sorbitol and 20g of Bacto - agar, yeast nitrogen base, 6.7g (W / O amino acid), 0.25g of Leu ^- feed, 4ml of 5 % Threonine, containing 40 ml of 50% glucose and 820 ml of water) and incubated at 30 ° C. for 3 to 5 days to obtain yeast colonies transformed with the recombinant plasmid.

Example 4

[Mass Expression of Human Growth Hormone in Yeast and Identification by SDS-Polyacrylamide Gel Electrophoresis]

A yeast strain transformed with the human growth hormone expression vector [pYLBC-A / G-UB-HGH] obtained in the usual practice 3, lacking 3 ml of leucine (yeucine without amino acids per liter of culture) 6.7 g of base (Yeast Nitrogen Base without amino Acids, Difco, USA) and 0.25 g of leucine-deficient amino acid mixture and 6% glucose) were incubated at 30 ° C. for 24 hours.

The final absorbance was about 25 OD at _650, where by centrifugation the amount corresponding to the OD ₆₅₀ of 10 was dissolved in 400㎕ buffer solution (10mM Tris HCl, pH 7.5, 1mM EDTA, 2mM PMSF, 8M urea), Glass beads with a diameter of 0.4 mm were added in the same volume and shaken vigorously to break the cell walls, thereby obtaining a yeast extract.

10 μl of the yeast extract was electrophoresed with 15% SDS polyacrylamide gel according to the method of Laemmli (Nature, 277, 680 (1970)), followed by staining with Coomassie brilliant blue R250. It is shown in the figure.

In Figure 4, lane 1 is the standard molecular weight of the protein (Bio-Rad, USA), 43000, 29000, 18400, 14300, 6200, and 2300 daltons from above, and lane 2 of yeast that does not contain the human growth hormone gene. Lanes 3 and 4 are extracts of the yeast containing the human growth hormone expression vector [pYLBC-A / G-UB-HGH] obtained in this example, respectively, 10 μl and 20 μl samples, and lane 5 is human growth. It is a standard sample of hormones.

As shown in FIG. 4, electrophoresis of the extract of the yeast containing the human growth hormone expression vector [pYLBC-A / G-UB-HGH] of lane 3 resulted in the absence of the human growth hormone gene of lane 2. Unlike conventional yeasts, human growth hormone linked to ubiquitin appears to have a dark band at the position of the human growth hormone standard, that is, the position corresponding to the molecular weight of 22,000 daltons, and the cleaved ubiquitin at the position of 5,500 daltons. It was confirmed that the first generation was produced by the enzyme in the yeast in the cell and mature human growth hormone.

As described above, the human growth hormone produced by the existing genetic engineering method requires a complicated process in which methionine remains and additionally removes it, unlike the hormone in the natural state, but the manufacturing cost is high. According to the human growth hormone expression vector [pYLBC-A / G-UB-HGH] according to the present invention, human growth hormone without methionine can be directly obtained. Human growth hormones can be prepared in the same way as human growth hormones.

Claims (8)

By adding primer 1 and primer 2 to the whole yeast nucleic acid, a ubiquitin gene containing 30 3′-terminal sequences of the effector gene having the restriction enzyme SacII BesBI site was synthesized, and this gene and the human growth hormone expression vector pYLBC-A / G-HGH was used as a template, and a polymerase chain reaction using primers 2 and 3 was carried out to synthesize a gene for the ubiquitin gene having SacII and BamHI sites, and then the gene and the human growth hormone expression vector pYLBC-A. Endogenous-Human Growth Hormone Fragments with BamHI and XhoI Sites from / G-HGH and XhoI and SacII Linked Synthetic Linkers Retaining Part of Human Growth Hormone and Ubiquitin Genes Recloned into Yeast Expression Carrier Human growth hormone expression vector pYLBC-A / G-UB-HGH. By adding primer 1 and primer 2 to the whole yeast nucleic acid, a ubiquitin gene containing 30 3′-terminal sequences of the effector gene having the restriction enzyme SacII BesBI site was synthesized, and this gene and the human growth hormone expression vector pYLBC-A / G-UB-HGH as a template, followed by polymerase chain reaction using Primer 2 and Primer 3 to synthesize an effector-ubiquitin gene having a SacII BamHI site, and then the gene and the human growth hormone expression vector pYLBC- Recloning of the endogenous-human growth hormone gene fragment having the BamHI and XhoI region obtained from A / G-UB-HGH and the XhoI and SacII-linked synthetic linker containing a part of human growth hormone gene and a part of ubiquitin gene were recloned into the yeast expression carrier. Method for producing a human growth hormone expression vector, characterized in that. The method of producing a human growth hormone expression vector according to claim 2, wherein the whole yeast nucleic acid is extracted from Saccharomyces cerevisiae AB110 cultured in YEPD medium consisting of 1% yeast extract, 2% peptone, and 2% glucose. The primer 1 of claim 2, wherein the primer 1 is a 53-mer, 5'-GTTTCGAATAAACACACATAAACAAACACCATGCAAATTTTCGTCAAAACTCT-3 ′, wherein the 3′-terminal base sequence of the A / G agonist gene is linked to 23 5′-terminal sequences of the ubiquitin gene. Method for producing a human growth hormone expression vector, characterized in that consisting of. The method according to claim 2, wherein the primer 2 is composed of 28-mer, 3'-GTGAACCACAACTCTGAGGCGCCACCA-5 ° complementary to the upper strand DNA of the ubiquitin gene 3'-terminus. The method according to claim 2, wherein primer 3 is composed of the same 24-mer, 5′-GCCACCATACCCACGCCGAAACAA-3 ′, identical to the top strand sequence above the gene of the human growth hormone expression vector pYLBC-A / G-UB-HGH. Method of producing a human growth hormone expression vector. 3. The XhoI-SacII linkage linker of claim 2 is a top strand 24-mer, 5'-GGTGGTTTCCCAACTATTCCACTC-3 'designed to be complementary to one of the SacII sites in the ubiquitin gene and the other to the XhoI site in the human growth hormone gene. And lower strand 31-mer, 3'-CGCCACCAAAGGGTTGATAAGGTGAGAGCTG-5 'method for producing a human growth hormone expression vector, characterized in that consisting of. The method for producing a human growth hormone expression vector according to any one of claims 2 to 7, wherein the human growth hormone expression vector is an expression vector pYLBC-A / G-UB-HGH.

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