RU2465315C1 - RECOMBINANT PLASMID DNA pPBS-St9 CODING SOMATOTROPIN POLYPEPTIDE, AND SACCHAROMYCES CEREVISIAE YEAST STRAIN FOR PRODUCING RECOMBINANT SOMATOTROPIN - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is recombinant plasmid DNA pPBS-St9 coding polypeptide having a sequence of the human growth hormone somatotropin of molecular weight 4.1 MDa (6266 base pairs), as well as the Saccharomyces cerevisiae strain BY4739 [leu2 ura3 lys2 prcl::LEU]/pPBS-St9 containing recombinant plasmid DNA pPBS-St9 that is a recombinant somatotropin producer.

EFFECT: invention may be used for producing the recombinant human growth hormone in treating dwarfism.

2 cl, 5 dwg, 2 ex

Description

Technical field

The invention relates to biotechnology, genetic engineering, the microbiological, medical and pharmaceutical industries and is an in vitro recombinant plasmid DNA that is designed to synthesize human somatotropin in Saccharomyces cerevisiae yeast cells, a method for constructing this plasmid DNA and a human yeast somatotropin producing strain containing this plasmid.

State of the art

Somatotropin (synonyms: growth hormone, human growth hormone, growth hormone, growth hormone growth hormone) belongs to the most studied pituitary hormones and belongs to the protein family, including growth hormone, prolactin, cholesterol. Somatotropin is synthesized in the anterior pituitary gland and in all mammalian species is a single peptide with a molecular weight of about 22,000 Da. Despite the high degree of homology of the sequences of growth hormones of various mammals, only human growth hormone or higher growth primates are active in human cells. Somatotropin molecule consists of 191 amino acid residues and contains two intramolecular disulfide bridges [Roskam W.G., Rougeon F. // Molecular cloning and nucleotide sequence of the human growth hormone structural gene. Nucl. Acids Res., 1979, v. 7, p. 305-320 .; Martial J.A., Hallewell R.A., Baxter J.D., Goodman H.M. // Human growth hormone: complementary DNA cloning and expression in bacteria. Science, 1979, v.205, No4406, p.602-607].

Somatotropin has an anabolic effect, has a positive effect on mineral metabolism, causes an increase in growth and body weight with dwarfism associated with growth hormone deficiency. Somatotropin is produced and secreted into the blood by specialized cells, mainly of the anterior pituitary gland - somatotrophs. The content of growth hormone in the human pituitary gland is more than an order of magnitude higher than the content of other hormones of this endocrine gland. Somatotropin is a multifunctional hormone. Being a specific stimulator of the growth of the body (skeleton and soft tissues), it is also involved in the regulation of all types of metabolism. The main defect in the development of the human and animal organism in conditions of growth hormone deficiency is a delay in bone growth. Excess growth hormone in a growing body can lead to gigantism, and in adults - to an abnormal increase in individual organs and tissues. The effect of growth hormone on bone growth is mediated through somatomedins, which are insulin-like growth factors of a polypeptide nature. Somatotropin biosynthesis and secretion are under complex control, including regulation of the hypothalamus hormones - somatostatin and somatoliberin, as well as some other hormones and metabolic products.

The scope of somatotropin is quite wide: a violation of the growth process in children with insufficiency of endogenous growth hormone, chronic renal failure in children, accompanied by stunted growth, Shereshevsky-Turner syndrome, osteoporosis, immunodeficiency syndromes, accompanied by weight loss.

Dosage forms of growth hormone (Genotropin (PFIZER MFG. BELGIUM, NV, manufactured by Vetter Pharma-Fertigung, GmbH & Co. KG; Humatrop (LILLY FRANCE, (France), Sayzen MERCK SERONO, SpA (Italy), Norditropin (NOVO NORDISK, A / S (Denmark), Biosoma, manufacturer BIOFA, Russian drug Rastan (PHARMSTANDART-UfaVITA, Russia), also known are nutropine or protropin (Genentech), Jintropin (GenSci, China), Dynatrope (Dynamic Development Laboratories Co., Ltd), Ansomone ( Ankebio, China), Getropin (Getropin, China), Hygetropin (China), Somatohorm (Biomed, Poland) and others.

A known method of producing growth hormone, consisting in its isolation from the tissues of the human pituitary gland [Simionescu L., Dimitriu V., Zamfir-Grigorescu D., Aman E., Terbancea M. // The simultaneous isolation of human pituitary hormones. I. Human growth hormone. Endocrinologie 1982 Oct.-Dec .; 20 (4): 273-83]. The disadvantages of the method are the difficulty in obtaining the source material and the risk of infection [Rappaport E.B. // latrogenic Creutzfeldt-Jakob disease. Neurology, 1987, Sep .; 37 (9): 1520-2].

A known method based on the production of recombinant growth hormone in transformed mouse cells of the line C 127 [Carter M.J., Facklam T.J., Long P.C., Scotland R.A. // Are continuous cell lines safe as substrates for human drugs and biologies? A case study with human growth hormone. Dev Biol Stand 1989; 70: 101-7]. With this approach, it is possible to obtain human growth hormone in the correct conformation with good physiological activity. The disadvantages of this method are the extremely low yield and the long cultivation time of the producing cells.

A promising approach for producing human growth hormone in significant quantities is the use of microorganisms as producers of this drug [Seeburg, et al. // Synthesis of growth hormone by bacteria. Nature, 1978, 276, 795-798; Seeburg et al. // Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone, 1978 Nature, 270, 486-494; Martial et al. // Human growth hormone: complementary DNA cloning and expression in bacteria, Science, 1979, 205, 602-607; Seeburg et al. // Efficient Bacterial Expression of Bovine and Porcine Growth Hormones, 1983, DNA 2: 37-45].

Currently, the bacterial strain Escherichia coli is known in the art, which is a producer of porcine somatotropin (RF Patents 2072393). A known method of producing somatotropin, including expression in Escherichia coli cells, which consists in a sufficiently fast biosynthesis of recombinant somatotropin by bacterial cells in the form of insoluble "inclusion bodies" [Olson KC, Fenno J., Lin N., Harkins RN, Snider C., Kohr WH, Ross MJ, Fodge D., Prender G., Stebbing N. // Purified human growth hormone from E. coli is biologically active. Nature 1981 Oct. one; 293 (5831): 408-11].

From US Pat. No. 5,795,745, a somatotropin producer based on the plasmid pHGH 107 is known that carries, under the control of two successive Lac promoters, a somatotropin gene consisting of a synthetic moiety (corresponding to amino acids 1-24) and a fragment of a natural gene (corresponding to amino acids 25-191). The use of Lac promoters, as well as almost completely (88%) of natural cDNA, including codons that are rarely found in Escherichia coli genes, leads to a significant decrease in the level of protein biosynthesis relative to the theoretically possible one.

In Russian patent 2233879, recombinant plasmid DNA pES1-6 was developed, which provides a high yield of recombinant growth hormone from E. coli bacteria. The disadvantage may be the use of the bacteria E. coli as a producer, since this organism is a conditional pathogen.

The use of eukaryotic expression systems, in particular yeast, allows for the correct post-translational modification of recombinant proteins; preparations obtained from yeast do not contain bacterial pyrogens and endotoxins, which can reduce the spectrum of side effects and significantly improve the quality of life of patients during long-term treatment. The closest in technical essence is US patent 4443539, in which the process of isolating bovine growth hormone from Saccharomyces cerevisiae yeast is patented.

For the production of recombinant human growth hormone, we propose the use of a yeast producer strain Saccharomyces cerevisiae that carries plasmid DNA with the growth hormone gene.

The implementation of the invention

The invention solves the problem of constructing a plasmid that determines the synthesis of recombinant protein, and create a highly productive bacterial producer strain that allows to obtain recombinant somatotropin in high yield and using simplified technology.

Stable maintenance of extrachromosomal genetic elements (plasmids) in a yeast cell depends on the presence of sequences in such elements that ensure DNA replication simultaneously with the replication of yeast chromosomes and uniform distribution of plasmids across daughter cells, as well as the possibility of selection of cells containing this plasmid.

The problem is solved by constructing a recombinant plasmid DNA pPBS-St9 encoding a polypeptide with a somatotropin sequence having a molecular weight of about 4.1 MDa (6266 bp). The plasmid was constructed on the basis of plasmid pRS316 [Sikorski and Hieter P. // A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, 1989, Genetics 122 (1): 19-27] standard centromeric shuttle vector, including a bacterial marker of ampicillin resistance and the origin of replication, a polylinker in the LacZ gene region, S. cerevisiae URA3 gene, ARS and CEN Saccharomyces cerevisiae sequences. The original plasmid pRS316 is commercially available, for example, in ATCC Number 77145 and can be ordered at http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATC CNum = 77145 & Template = vectors. The recombinant plasmid DNA pPBS-St9 includes the PHO5 gene promoter (inserted at SacI / SacII sites position 1998-2533, insert size 536 bp), DNA fragments encoding the 24 N-terminal amino acids of human interleukin hIL-1beta (Ser5- Ala28), inserted at the SacII / Xba1 sites (position 2540-2611, insert size 72 bp), site for cleavage with endopeptidase Kech2 (Lys-Arg), 191 a.k. growth hormone inserted at the Xba / BamHI sites (position 2617-3192, insert size 576 bp), after which there is a transcription termination signal for the yeast CYC1 gene inserted at the HindIII / BamHI sites (position 3198-3460, insert size 262 p. about.).

To regulate the expression of the human somatotropin gene, we used the PHO5 gene promoter containing regions that activate transcription in the absence of inorganic phosphate in the culture medium, as well as the region of transcription initiation. The PHO5 gene promoter is one of the most powerful yeast promoters. The level of expression of genes under the control of the PHO5 promoter is effectively regulated by exogenous inorganic phosphate. At a concentration of inorganic phosphate in the culture medium of 1 g / l, only a basal expression level is observed. This allows you to regulate the synthesis of growth hormone in yeast cells. Thus, the proposed system allows to produce a large amount of yeast biomass under optimal conditions (on a medium containing inorganic phosphate) and then, after changing the cultivation conditions (replacing a medium containing inorganic phosphate with a medium without inorganic phosphate), to achieve a high yield in a short time the final product is somatotropin protein.

The construction of the recombinant gene encoding human somatotropin was carried out on the basis of plasmid pRS316 4887 bp [Sikorski and Hieter P. // A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, 1989, Genetics 122 (1): 19-27]. The synthesized recombinant gene is inserted into the pRS316 plasmid at the XbaI / BamHI sites. An artificial gene encoding somatotropin flanked by restriction enzyme sites XbaI and BamHI is obtained by enzyme-chemical synthesis of a set of oligonucleotide fragments, followed by their assembly and amplification using polymerase chain reaction (PCR). Before ligation to generate sticky ends, the amplification and vector plasmid is treated with restriction enzymes XbaI and BamHI. The ligase mixture is used to transform competent E. coli DH5α cells. The selection of positive clones is carried out using PCR using specific primers, followed by restriction analysis of the isolated plasmid DNA with restriction enzymes PstI and XbaI. The structure of the gene encoding the recombinant somatotropin is determined by Senger sequencing on an ABI 377 instrument. It should fully correspond to the nucleotide sequence of the original somatotropin artificial gene (Fig. 1).

Due to the high variability of the codon usage index in yeast, deviations in the presented nucleotide sequence of the somatotropin gene are possible. The inventive step extends to sequences with homology from 90% to 100% of the present invention.

Recombinant plasmid DNA pPBS-St9 encoding a somatotropin polypeptide is characterized by the following features:

- has a molecular weight of about 4.1 Mda (6266 bp);

- encodes a somatotropin polypeptide;

- consists of: XbaI / BamHI DNA fragment of plasmid pRS316-St containing the synthetic somatotropin gene, plasmid pRS316, including the bacterial marker of ampicillin resistance and the origin of replication, polylinker in the LacZ gene region, S. cerevisiae URA3 gene, ARS and CEN sequences S. cerevisiae; the sequence flanked by the SacI / XbaI sites of human interleukin hIL-1beta from the plasmid YEpsec1-hI1; The transcription termination signal of the yeast CYC1 gene from the plasmid pCM251 is inserted at the HindIII-BamHI sites, the PHO5 promoter is amplified with the chromosomal DNA of strain 74-D694 and then inserted at the SacII-SacI sites in the vector. Unique recognition sites PstI and XbaI give fragments of 2240 and 6022 bp when treated with appropriate restriction enzymes.

The plasmid contains a marker gene for selection - the URA3 gene. A strain carrying a complete deletion of the ura3 gene acts as a recipient for this plasmid. The ability to synthesize uracil will be characteristic only of cells carrying the plasmid; therefore, only stable transformants will grow on a synthetic medium without exogenous amino acids and nitrogen bases. The strain used was the yeast strain Saccharomyces cerevisiae BY4739 [leu2 ura3 lys2 prc1 :: LEU2].

To obtain a producer strain of recombinant somatotropin, plasmid DNA pPBS-St9 is used to transform competent BY4739 prc1 :: LEU2 yeast cells and select clones that maintain the level of biosynthesis of the recombinant polypeptide at least 20% of the total cellular protein for at least six consecutive passings. To do this, clones of transformed plasmid DNA are grown in rich YPD medium with the addition of KH ₂ PO ₄ at a concentration of 1 g / l for 12-14 hours, a new portion of the nutrient medium is inoculated in a ratio of 1: 100, and the culture is incubated until the optical density is A ₆₀₀ = 1, washed from the nutrient medium, add rich YEPD medium and incubate for another 3-6 hours. Obtaining recombinant somatotropin from producer cells includes the following steps: separating the culture fluid; solubilization and restoration of the target protein, its refolding and final purification.

The original strain BY4739 of the MAT genotype leu2 lys2 ura3 is freely sold by Gentaur, see link http://www.clonagen.com/clonagen/c1106c02-bbb5-45a3-a8bf-b011aabfle4c/yeast_parental_strains_by4742_product of this invention can be used for implementation of .

The resulting producer strain Saccharomyces cerevisiae BY4739 prcl :: LEU2 / pPBS-St9 is characterized by the following features.

Morphological signs: round cells.

Cultural traits: when growing on an agarized YEPD medium, the colonies are round, smooth, white, the edge is even. When growing on a liquid medium, YEPDs form an intense smooth haze.

Physico-biological characteristics: cells grow at a temperature of 4 ° C to 40 ° C with an optimum pH of 5.5-6.0. As a source of nitrogen, both mineral salts in the ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. As a carbon source, glucose, glycerin, and sucrose are used.

The advantages of the present invention are, firstly, in the use of the somatotropin gene in chemical-enzymatic synthesis of the widest possible set of codons that are optimal for the production of protein in Saccharomyces cerevisiae, the location of which in the synthetic gene eliminates the possibility of the formation of extended "hairpins" that potentially inhibit mRNA broadcast. Figure 1 shows the nucleotide sequence of the XbaI / BamHI fragment of the plasmid pPBS-St9, containing the recombinant human somatotropin gene, optimized for expression in S. cerevisiae yeast, combined with the interleukin gene fragment and the endopeptidase site Kex2; the corresponding amino acid sequence is shown in figure 2.

Secondly, the use of optimal regulatory elements that control its expression for biosynthesis of the recombinant protein: the regulated PHO5 promoter, the highly efficient CYC1 transcription terminator, the hIL-1beta human interleukin gene sequence for ejecting (excreting) the protein into the intercellular space, which greatly simplifies protein purification allows to achieve higher purity in comparison with a protein having intracellular localization.

Thirdly, the advantage of the proposed producer strain is the use of yeast with a deletion of vacuolar peptidase C, which ensures the stability of a foreign protein in yeast cells by inhibiting the proteolytic cleavage of the synthesized de novo recombinant growth hormone (somatotropin).

The cells of the strain S. cerevisiae BY4739 prc1 :: LEU2 / pPBS-St9 are superproducer. Upon induction of the PHO5 promoter, an effective biosynthesis of recombinant growth hormone occurs, which accumulates in the cells in an amount of at least 20% of the total yeast protein.

Figure 1 shows the nucleotide sequence of the XbaI / BamHI fragment of the plasmid pPBS-St9, containing the recombinant human somatotropin gene, optimized for expression in S. cerevisiae yeast, combined with the interleukin gene fragment (marked in bold) and the endopeptidase site Kex2 (underlined) ; figure 2 shows the amino acid sequence of the XbaI / BamHI fragment of the plasmid pPBS-St9 containing the recombinant human somatotropin gene, optimized for expression in S. cerevisiae yeast, combined with the interleukin gene fragment (marked in bold) and the endopepididase site Kex2 (marked with underscore) ; figure 3 presents the physical map of the plasmid pPBS-St9; figure 4 - nucleotide sequence of the promoter of the PHO5 gene; figure 5 - nucleotide sequence of the transcription terminator of CYC1.

It should be understood that a person skilled in the art can, without further experimentation, taking into account the present description, apply the present invention in practice in its entirety. Therefore, the examples given in this context are presented only to illustrate the invention, not reducing the scope of claims.

EXAMPLE 1

Construction of recombinant plasmid DNA pPBS-St9. The plasmid pPBS-St9 for expression of somatotropin includes the PHO5 gene promoter, DNA fragments encoding the 24 N-terminal amino acids of human interleukin hIL-1beta (Ser5-Ala28), a site for cleavage by endopeptidase Kex2 (Lys-Arg), 191 a.a. growth hormone, after which there is a signal for termination of transcription of the CYC1 gene of yeast.

The recombinant plasmid DNA pPBS-St9 was constructed on the basis of plasmid pRS316 [Sikorski and Hieter P. // A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, 1989, Genetics 122 (1): 19-27] - a standard centromeric shuttle vector, including a bacterial marker of ampicillin resistance and the origin of replication, a polylinker in the LacZ gene region, S. cerevisiae URA3 gene, ARS and CEN Saccharomyces cerevisiae sequences. Plasmid YEpsec1-hI1 [Baldari C, Murray JAH, Ghiara P, Cesareni G, Galeotti CL // A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1β in Saccharomyces cerevisiae was used as a source of the human interleukin hIL-1beta sequence , 1987, EMBO J 6: 229-234]. The sequence was amplified using a pair of primers:

The resulting fragment was then inserted into the vector at SacII-XbaI restriction sites.

The yeast CYC1 gene transcription termination signal was cut from the plasmid pCM251 [Belli G, Gari E, Piedrafita L, Aldea M, Herrero E. // An activator / repressor dual system allows tight tetracycline-regulated gene expression in budding yeast, 1998, Nucleic Acids Res. 26 (4): 942-7.] At the HindIII-BamHI restriction sites and is integrated into the vector at the same sites.

The PHO5 gene promoter was obtained by PCR with a pair of primers

with yeast chromosomal DNA (strain BY4739 and then inserted at the SacII-SacI sites into the vector).

The recombinant human somatotropin gene was obtained by chemical-enzymatic synthesis and cloned into pRS316 recombinant plasmid DNA at XbaI-BamHI sites.

Construction of recombinant plasmid DNA pRS316-St9.

The nucleotide sequence corresponding to the somatotropin gene is obtained by chemical-enzymatic synthesis. To do this, the theoretically calculated DNA sequence is divided into overlapping fragments of about 50 bp in size. The chemical synthesis of oligonucleotides corresponding to these fragments is carried out by the solid-state phosphoamidite method using, for example, the ASM-900 DNA synthesizer (BIOSSET, Novosibirsk) with the oligonucleotide chain being expanded from the 3'-end to the 5'-end using protected phosphamidites - 5 '-dimethoxytrityl-N-acyl-2'-deoxynucleoside-3'-O- (β-cyanethyl-diisopropylamino) phosphites activated by tetrazole. The synthesis is carried out on a scale of 0.5-0.7 μmol, using porous glass (pore size 500 A) as a carrier, to which the first nucleoside unit (load 20-30 μmol / g) is connected via a 3'-succinate bond. The resulting oligonucleotides are subjected to 5'-terminal phosphorylation using T4 polynucleotide kinase (Fermentas, Lithuania). For this, oligonucleotides in an amount of 20 pmol are mixed with the enzyme in an amount of 10 units. in a buffer solution containing 50 mM Tris-HCl (pH 7.6 at 25 ° C), 10 mM MgCl ₂ , 5 mM dithiotreite, 1 mM spermidine, 0.1 mM ATP and 0.1 mM EDTA. The reaction is carried out for 30 minutes, the polynucleotide kinase is inactivated by heating to 65 ° C for 10 minutes.

Phosphorylated oligonucleotides are mixed in an equimolar ratio in 50 μl of a buffer containing 20 mM Tris-HCl, pH 7.56, 10 mM MgCl ₂ , 0.2 mM rATP, 10 mM dithiotreite, heated to 65 ° C, slowly cooled to 37 ° C for an hour and add 10 units. ac. T4 DNA ligases. The DNA ligation reaction is carried out for 4 hours at 37 ° C. 0.1 μl of the resulting solution is used as a template in the polymerase chain reaction (PCR) in the presence of thermostable Pfu DNA polymerase and specific primers

25 amplification cycles (95 ° C, 20 s; 62 ° C, 40 s; 72 ° C, 60 s) are carried out to synthesize a full-sized DNA fragment containing the somatotropin gene sequence flanked by BamHI and XbaI restriction enzyme recognition sites. The amplification product is hydrolyzed with restriction enzymes BamHI and XbaI, purified by electrophoresis in 5% acrylamide gel, a DNA strip of about 600 bp. isolated from the gel by electroelution and precipitated DNA from the solution with ethanol.

To prepare pRS316-St plasmid DNA with a recombinant somatotropin gene, the original pRS316 vector plasmid DNA (3 μg, 1 pmol) is treated with 40 μl of 2xY buffer (33 mM Tris-acetate, pH 7.9, 10 mM Mg-acetate, 66 mM K -acetate 1, 0.5 mm DTT, 0.1 mg / ml BSA) with restriction enzymes BamHI and XbaI (10 units per act) for 1 h at 37 ° C. The resulting DNA fragment of about 4.8 kbp after electrophoretic separation in a 1% agarose gel, it is isolated from the gel by electroelution and DNA is precipitated from the solution with ethanol. 1 μg of the obtained vector fragment is ligated with 2 pmol of a 576 bp BamHI / XbaI fragment containing the synthetic recombinant somatotropin gene and endopeptidase site in 10 μl of buffer (20 mM Tris-HCl, pH 7.56, 10 mM MgCl ₂ , 0.2 mM rATP, 10 mM dithiothreitis) using 10 units. Act. T4 DNA ligases for 12 hours at 10 ° C.

At the final stage, the BamHI / XbaI fragment of plasmid pRS316-St, containing the recombinant somatotropin gene and endopeptidase site, was ligated into the resulting plasmid. Figure 3. presents a physical map of plasmid DNA pPBS-St9.

The obtained ligase mixture transform cells of a strain of DH5a Escherichia coli. To obtain competent Escherichia coli cells, bacteria are grown in 100 ml of LB medium at 37 ° C until the culture reaches a cell suspension density of 0.4-0.6 units. optical density at a wavelength of 550 nm. The cell suspension is cooled in an ice bath, centrifuged at 5000 rpm for 10 minutes at 4 ° C. Cells are suspended in 100 ml of 10 mm sodium chloride, collected by centrifugation under the same conditions. Then the cells are suspended in 50 ml of 75 mm calcium chloride, kept in an ice bath for 40 minutes, precipitated by centrifugation under the same conditions and suspended in 1 ml of 75 mm calcium chloride. Glycerin is added to a suspension of competent cells to a final concentration of 15%, aliquoted and stored at -70 ° C.

At the stage of transformation of competent cells with the plasmid pPBS-St9, the suspension of competent cells is thawed in an ice bath, 1 μl of ligase mixture is added and incubated in an ice bath for 40 minutes. Next, the cells are subjected to heat shock at 42 ° C for 2 minutes, after which they are incubated in 1.5 ml of LB medium at 37 ° C for 1 hour. Cells were harvested by centrifugation at 5000 rpm for 10 minutes and plated on Petri dishes with LB medium containing 2% agar and 50 mg / L ampicillin. Cups are incubated at 37 ° C for 12-16 hours. The primary selection of clones containing the desired plasmid is carried out by the method of "PCR from clones" using specific primers:

Plasmid DNA was isolated from the grown individual clones of the transformants using the procedure (micromethod) described below, except that Escherichia coli cells were grown in 10 ml of LB and, accordingly, the volume of all solutions was reduced by a factor of 100. In addition, instead of a centrifugation step in the density gradient of cesium chloride, DNA is treated with pancreatic RNase. For this, nucleic acids precipitated with isopropyl alcohol are dissolved in 100 μl of TE buffer, 10 μl of RNase solution (1 mg / ml) is added and incubated for 30 minutes at 37 ° C.

To obtain preparative quantities of the plasmid, a maximode is used. For this, Escherichia coli bacteria cells containing the pPBS-St9 plasmid are grown overnight in 1 L of LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) containing ampicillin at a concentration of 50 mg / L. Cells are collected by centrifugation at 5000 rpm for 10 minutes at 4 ° C, the pellet is suspended in 20 ml of 25 mm Tris-chloride buffer (pH 8.0) containing 10 mm EDTA and 50 mm glucose. 30 mg of lysozyme was added to the suspension and incubated for 10 minutes at room temperature. Then add 40 ml of a 0.2 M NaOH solution containing 1% sodium dodecyl sulfate, mix gently and incubate for 10 minutes at room temperature. The solution is neutralized by adding 30 ml of 3 M sodium acetate (pH 5.0) and incubated for 10 minutes at 4 ° C. After that, centrifuged at 14000 rpm for 40 minutes at 4 ° C. 0.6 volumes of isopropyl alcohol are added to the supernatant, kept for 20 minutes at room temperature and centrifuged at 14000 rpm for 20 minutes at 20 ° C. The resulting precipitate is washed with 70% ethyl alcohol, dried in vacuum and dissolved in 4 ml of distilled water. Then 4.2 g of cesium chloride and 0.36 ml of ethidium bromide solution (10 mg / ml) are added. The resulting solution was incubated for 1 hour at 4 ° C, then centrifuged at 15,000 rpm for 15 minutes. The supernatant is centrifuged at 70,000 rpm for 16 hours in a TL-100 centrifuge ("Beckman"). After centrifugation, a strip of plasmid DNA is selected (the lower of the two bands fluorescent in ultraviolet light), ethidium bromide is extracted twice with an equal volume of iso-amyl alcohol, diluted twice with distilled water, and plasmid DNA is precipitated with two volumes of ethyl alcohol and 1/15 volume of 3 M acetate sodium (pH 5.0). The precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, washed with 70% ethyl alcohol and dissolved in 0.5-1 ml of TE buffer (10 mm Tris-chloride buffer, pH 8.0, containing 1 mm EDTA). The concentration of plasmid DNA is determined by the absorption of the solution at a wavelength of 260 nm. The purity of the drug is controlled by electrophoresis in a 0.7% agarose gel in TBE buffer (0.1 M Tris-borate buffer, pH 8.3, containing 1 mM EDTA).

Plasmid DNA was preparatively isolated from the cells of the identified clone by the above methods and used to transform yeast cells, as described in example 2.

EXAMPLE 2

Recombinant growth hormone producer strain.

To obtain a yeast strain Saccharomyces cerevisiae, a producer of somatotropin, we used strain BY4739 of the genotype MAT leu2 lys2 ura3, with inactivated vacuolar peptidase C and two markers of auxotrophy, transformed with plasmid pPBS-St9.

To obtain a deletion of vacuolar peptidase C, we performed PCR of chromosomal DNA of strain BY4739 with primers

The first 40 nucleotides of these primers are homologous to the sequences flanking the PRC1 gene on the chromosome, the remaining 20 coding sequences of the LEU2 gene. The result is a fragment containing the full-sized LEU2 gene, flanked by 3 'and 5' PRC1 gene sequences. Transformation of the [ura3 leu2 lys2] strain with such a fragment leads to homologous recombination over flanking sequences, the appearance of Leu + clones in which the PRC1 gene is replaced by the LEU2 gene.

Subsequently, we transformed the strain [leu2 ura3 lys2 prc1 :: LEU2] with our plasmid with the somatotropin gene and constructed Ura + clones carrying the plasmid.

For this, yeast cells are grown in 100 ml of YEPD medium until the culture reaches an optical density corresponding to 2-4 units. absorption at a wavelength of 600 nm. The cells are washed twice with sterile water, after which they are suspended in 0.3 ml of a 100 mM lithium acetate solution and incubated at 30 ° C. for 30 minutes. To 50 μl of the obtained cell suspension, 0.1-1 μg of plasmid DNA, 50 μg of salmon sperm DNA previously denatured by heating (10 minutes at 100 ° C) and 0.3 ml of a solution of 100 mM lithium acetate containing 40% PEG- polyethylene glycol are added 4000. The sample is then incubated for 30 minutes at 30 ° C and 20 minutes at 42 ° C, placed for 15 seconds in an ice bath and centrifuged for 10 seconds at 10,000 rpm. Cells are suspended in 1 ml of sterile water and plated on solid MD medium. Clones of transformants grow after 4-6 days.

For the analysis of growth hormone production by transformant cells, they are grown in 50 ml of PEP liquid medium until the stationary growth phase. Cells are harvested by centrifugation at 3000 rpm for 10 minutes, washed with water, suspended in 1 ml of 50 mM Tris-chloride buffer (pH 7.5) containing 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 g of glass beads (0.5 mm) and destroyed in the Braun disintegrator for 1 minute at 4 ° C. The resulting homogenate is centrifuged at 10,000 rpm for 10 minutes, the precipitate is washed with a 10-fold volume of the same buffer and suspended in 1 ml of 50 mm sodium phosphate buffer (pH 7.0) containing 2% sodium dodecyl sulfate and 5% 2- mercaptoethanol, and incubated for 5 minutes in a boiling water bath. At the end of the incubation, the sample is centrifuged for 15 minutes at 12000 rpm and the somatotropin content is determined in the supernatant by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent hybridization with specific antibodies to human somatotropin. Protein separation is carried out in a 15% polyacrylamide gel in a standard buffer system (electrode buffer: 25 mm Tris, 192 mm glycine, 0.1% sodium dodecyl sulfate, pH 8.3; gel buffer: 375 mm Tris-chloride buffer, pH 8, 8).

In parallel, the separation of proteins of the control (not transformed by plasmid) strain BY4739 prcl :: LEU2, grown and homogenized under identical conditions, is carried out.

Carbonic anhydrase, trypsin inhibitor, myoglobin, and Sigma lysozyme are used as molecular weight standards. At the end of electrophoresis, the proteins are renatured, keeping the gels for 15 minutes in 10 mM Tris-chloride buffer (pH 7.5) containing 4 M urea, 20 mM EDTA. The proteins are then transferred onto a nitrocellulose membrane in 25 mM Tris-192 mM glycine buffer (pH 8.3) containing 20% methyl alcohol, at 30-40 V, for 1.5 hours using a Bio-Rad instrument. Next, the membrane was kept in TBST buffer (10 mM Tris-chloride buffer (pH 8.0), 150 mM sodium chloride, 0.05% tween-20, 1% bovine serum albumin) for 2 hours at 26 ° C. The membrane is then placed in the same buffer containing 1000 times diluted rabbit polyclonal antibodies to human growth hormone ("Abeam") and incubated for 2 hours at 26 ° C. The membrane is washed three times with TBST buffer and incubated for 1 hour at 26 ° C with a conjugate of species-specific antibodies to rabbit immunoglobulins and horseradish peroxidase ("Abcam") diluted 7000 times. After washing the membrane with PBST buffer (58 mM disubstituted sodium phosphate, 17 mM monosubstituted sodium phosphate, 68 mM sodium chloride, 0.1% Tween-20), it was placed in a solution of peroxidase substrates: 0.02% DAB (3, 3- diaminobenzidine tetrahydrochloride), 0.006% hydrogen peroxide in 10 mM Tris-chloride buffer, pH 7.5. In parallel, the gels are stained with a 0.15% solution of Coomassie G250 in 25% isopropanol and 10% acetic acid and washed in 10% acetic acid. When comparing the protein spectrum of the two strains in strain BY4739 prc1 :: LEU2 / pPBS-St9, an additional protein band appears that gives a positive reaction with antibodies to human growth hormone with a molecular weight of 22 kDa, which corresponds to the molecular weight of growth hormone. The level of synthesis of recombinant growth hormone is determined by comparing the staining intensity of a strip of this protein with a strip of standard growth hormone. According to the data obtained, yeast cells of strain BY4739 prc1 :: LEU2 / pPBS-St9 synthesize intracellularly about 50 mg of growth hormone per liter of yeast culture. When analyzing the suppression of the cytopathic activity of the virus of vesicular stomatitis in a culture of human fibroblasts using standard methods [M. G. Tovey et al. // Antiviral Activity of Bovine Interferons on Primate Cells, 1977, J. Gen. Virol., 36, 341-344] it is found that the preparation of growth hormone synthesized by the yeast strain Saccharomyces cerevisiae BY4739 prc1 :: LEU2 / pPBS-St9 is biologically active. The activity of the drug is 20-40 million units / liter.

Summarizing the above, it can be concluded that the obtained strain of yeast Saccharomyces cerevisiae BY4739 prc1 :: LEU2 / pVBS-St9 synthesizes human growth hormone in an amount sufficient for its purification on a laboratory and industrial scale. As a result of this purification, somatotropin preparations of therapeutic value suitable for use in medicine can be obtained.

The yeast strain Saccharomyces cerevisiae BY4739 [leu2 ura3 lys2 prc1 :: LEU] / pPBS-St9, was deposited in the Peterhof genetic collection of microorganisms under the number C-016 - producer of recombinant human growth hormone.

Claims (2)

1. Recombinant plasmid DNA pPBS-St9, encoding a polypeptide with a sequence of human growth hormone somatotropin, having a molecular weight of 4.1 MDa (6266 bp); consisting of the SacI / HindIII DNA fragment of the plasmid pRS316 containing the bacterial marker of ampicillin resistance and the origin of replication, the polylinker as part of the LacZ gene region, unique restriction endonuclease recognition sites located at the following distance to the right of the PstI site: XbaI - 2236 bp , URA3 gene, ARS and CEN sequences of Saccharomyces cerevisiae, as well as the PHO5 gene promoter sequence inserted at the SacI / SacII sites at position 1998-2533 (insert size 536 bp) inserted into the original plasmid DNA, DNA fragments encoding 24 N -term amino acids hIL-1beta (Ser5-Ala28) human interleukin inserted at sites SacII / XbaI (position 2540-2611, insert size 72 bp) cleavage site for endopeptidase Keh2 (Lys-Arg), aa 191 growth hormone inserted at the Xba / BamHI sites (position 2617-3192, insert size 576 bp), after which the signal sequence for the transcription of the yeast CYC1 gene is inserted, inserted at the HindIII / BamHI sites (position 3198-3460, insert size 262 p .about.). 2. The strain Saccharomyces cerevisiae BY4739 [leu2 ura3 lys2 prc1 :: LEU] / pPBS-St9 containing the recombinant plasmid DNA pPBS-St9 according to claim 1, is a producer of recombinant somatotropin.

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