RU2460795C1 - Method for microbiological synthesis of secreted human somatotropin and yeast strain saccharomyces cerevisiae - secreted human somatotropin producer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is produced is the yeast strain Saccharomyces cerevisiae able to produce secreted human somatotropin. Said strain contains a promoter contolled DNA sequence coding mature human somatotropin fused with a leader peptide in the same reading frame. The leader peptide includes a double pro-site of α-factor of yeast Saccharomyces cerevisiae. It also can contain a triple pro-site of α-factor of yeast Saccharomyces cerevisiae, or a double pro-site of HSP150 protein of yeast Saccharomyces cerevisiae, or a combination of said pro-sites. A method for producing human somatotropin provides cultivation of the human somatotropin producer strain.

EFFECT: use of the invention provides higher end product yield.

2 cl, 1 dwg, 8 ex

Description

The invention relates to the field of biotechnology and relates to a method for the synthesis of secreted human growth hormone by Saccharomyces cerevisiae yeast cells.

Increasing the production of medical and technical proteins by increasing the efficiency of their secretion in the cells of microorganisms is one of the priority areas of research in the field of biotechnology. Microorganisms used for these purposes include Saccharomyces cerevisiae yeast.

By secreted proteins we mean such soluble target proteins, the biosynthesis of which in S. cerevisiae yeast cells is accompanied by their secretion into the extracellular medium.

It is known that the efficiency of secretion in S. cerevisiae yeast cells depends on the choice of leader polypeptides (leader regions) that direct protein secretion [Romanos et al., 1992, Yeast 8: 423-488]. A necessary part of the leader polypeptide is the signal peptide (pre-region), an additional part is the pro-region. Each of the parts of the leader polypeptide that make up the secreted protein at the stage of its biosynthesis is removed from its composition during secretion as it performs its function.

The main function of the signal peptide is to facilitate the process of protein translocation, i.e. the secreted protein intersects the membrane layer separating the region of the cell in which the protein is synthesized from the region of the reticulum, in which the secreted protein is folded and its post-translational modification. The signal peptide is removed from the composition of the secreted protein directly in the process of translocation. The pro-region can fulfill a twofold function: on the one hand, the pro-region provides elongation of proteins, especially short proteins, affecting their translocation. The second known function of the pro-region is realized at the stage of protein transport from the reticulum and consists in the interaction of the sequence of the pro-region with transport receptors, which mediate the incorporation of secreted proteins into secretory granules in which secreted proteins move within the cell. At one of the final stages of intracellular transport in the Golgi complex, enzymatic cleavage (processing) of the pro-region from the mature part of the secreted protein takes place, which is then sent to the plasma membrane and exit the cell.

In S. cerevisiae yeast, leader regions of yeast secreted proteins are commonly used as leader regions. One of the most widely used leader regions of yeast origin used for secretion of heterologous proteins in S. cerevisiae cells is the leader pre-pro region of the α-factor precursor (MFα-1), a sex factor secreted by α-type mating yeast cells. The effectiveness of this leader region has been demonstrated many times, including in relation to the secretion of proteins such as insulin-like growth factor I [Bayne et al., 1988, Gene, 66: 235-244], granulocyte colony-stimulating human factor [Ernst, 1988, DNA, 7 : 355-360], human and chicken lysozymes [Oka et al., 1999, Biosci. Biotechnol. Biochem., 63: 1977-1983; Hashimoto et al., 1998, Protein Engineering, 11: 75-77].

Another well-known secretory leader of yeast origin is the pre-pro-region of the S. cerevisiae HSP150 glycoprotein, whose positive effect on the secretion of heterologous proteins in yeast cells has been established, for example, for β-lactamase [Simonen et al., 1994, J Biol Chem, 269: 13887-92] and the extracellular domain of rat nerve growth factor receptor [Simonen et al., 1996, Yeast, 12: 457-66].

An example of a sequence of artificial origin that effectively performs the functions of a leader polypeptide in yeast cells is a fragment of protein interleukin-1-beta [Lee et al., 1999, Biotechnol Prog, 15: 884-890].

A number of modifications of pro-regions of leader polypeptides have been found that provide an improvement in their processing [Kjeldsen et al., 1996, Gene, 170: 107-112] and secretory activity [Kjeldsen, 2000, Appl Microbiol Biotechnol, 54: 277-286]. In particular, an artificially engineered pro-peptide is known that provides a 4-fold increase in human insulin secretion compared to the standard pro-region of the yeast α-factor [Kjeldsen, 2000, Appl Microbiol Biotechnol, 54: 277-286].

Human growth hormone (growth hormone) belongs to the group of anabolic hormones. Its insufficiency in the human body leads to grave consequences - dwarfism, accompanied in most cases by degenerative changes in the skeleton and muscles, infertility, and sometimes mental retardation. The physiological effect of growth hormone includes an increase in muscle growth and strength, stimulation of bone and cartilage tissue formation, increased fat breakdown, and improved cardiac activity [Hedge et al., 1987, Clinical endocrine physiology. W.B.Saunders company, 1987]. Somatotropin also has a pronounced anti-atherogenic effect (it increases the level of high density lipoproteins and lowers the level of low density lipoproteins. It has been shown that people with growth hormone deficiency have an increased risk of cardiovascular mortality (Materials of the III All-Russian Scientific and Practical Conference "Actual Problems of Neuroendocrinology", Moscow, October 6-7, 2003).

A method for constructing S. cerevisiae SCR-H1 and SCR-H2 strains, which are stable producers of secreted recombinant human growth hormone, is described. The cells of these strains contain several copies of expression cassettes, including the somatotropin gene, integrated into various chromosomal loci, which ensures their stable inheritance. In the expression cassettes, the human somatotropin gene is under the control of the GAL1 promoter S. cerevisiae. Protein secretion is directed either exclusively by the leader pre-pro-region of the yeast α-factor (in cells of the SCR-H1 strain), or two leaders are used simultaneously, which are alternative to each other: in terms of constructs, somatotropin secretion is directed by the leader pre-pro-region yeast α-factor, and in others, a hybrid leader region containing a modified signal peptide of yeast α-factor and the pro-region of S. cerevisiae HSP150 protein (in cells of strain SCR-H2). The level of growth hormone secretion is 100 mg / l and 150 mg / l, respectively [RU 2009145487].

The task of the claimed group of inventions is to expand the arsenal of methods for microbiological synthesis of secreted human growth hormone by Saccharomyces cerevisiae yeast cells.

The problem is solved by

- development of a method for microbiological synthesis of secreted human growth hormone by Saccharomyces cerevisiae yeast cells, which cultivates a producer strain of secreted human growth hormone containing a DNA sequence encoding a mature human growth hormone fused in the same reading frame with a leader polypeptide comprising a signal peptide and a combination of two or three sequentially arranged pro-regions, each of which stimulates the secretion of human growth hormone in the yeast Saccharomyces cerevisiae, per hour doubled pro-region of S. cerevisiae yeast α-factor, tripled pro-region of S. cerevisiae yeast α-factor, doubled pro-region of S. cerevisiae yeast HSP150 protein or pro-region of S. cerevisiae yeast HSP150 protein and pro-region S. cerevisiae yeast α-factor;

- designing a strain of yeast Saccharomyces cerevisiae VKPM Y-3580 - producer of secreted human growth hormone.

The method in General

Inoculum culture of one of the strains of yeast Saccharomyces cerevisiae - a producer of secreted human growth hormone containing a DNA sequence encoding a mature human growth hormone fused in the same reading frame with a leader polypeptide comprising a signal peptide and a combination of two or three consecutively located pro-regions, each of which stimulates secretion of growth hormone in yeast Saccharomyces cerevisiae, obtained by culturing the strain for 16-40 hours at a temperature of 22-32 ° C on an orbital shaker 100-350 rpm in medium the following composition in wt%: peptone 1-3;. yeast extract 0.5-3; glucose 1-3, water - the rest, pH - natural.

A fermenter (0.5-1000 L) containing a fermentation medium of the following composition, in wt.%: Peptone 1-3; yeast extract 0.5-3, glucose or sucrose 1-4, water - the rest, pH - natural. The amount of seed culture introduced into the fermenter is 3-15% of the volume of the fermentation medium.

Fermentation is carried out at a temperature of 22-32 ° C, aeration of 0.2-1.5 rpm / min and the mixing speed of the culture of 200-1500 rpm. After 16-30 hours after sowing the fermenter begin feeding with an aqueous solution of the following composition (in grams per liter): peptone 20-100; yeast extract 20-100, glucose or sucrose 100-500, with a speed of 1-10 ml / l / h and set the pH-stating of the culture in the pH range 5.5 - pH 7.2. The total duration of fermentation is 48-96 hours.

The method allows to synthesize secreted human somatotropin in an amount of 1-2 g / l of culture fluid, to determine the amount of which the method of polyacrylamide gel electrophoresis and enzyme immunoassay are used [EN 2009145487].

Construction of a strain of Saccharomyces cerevisiae - producer of secreted human growth hormone.

Stage 1 - construct variants of genes of new leader polypeptides that comprise protein sequences encoding an effective signal peptide and a combination of successively located pro-regions, each of which stimulates the secretion of human growth hormone in Saccharomyces cerevisiae yeast, in particular, doubled α-pro-region S. cerevisiae yeast factor, or triple pro-region of S. cerevisiae yeast α-factor, or doubled pro-region of S. cerevisiae yeast HSP150 protein, or S. cerevisiae yeast protein pro-region and yeast α-pro-region Ms. S. cerevisiae.

By combination is meant the animation of a unique pro-region or a combination of different pro-regions. The new C-terminal leader polypeptides contain a unique KEX2 proteinase recognition site.

Stage 2 - construct variants of yeast expression vectors, each of which contains a gene encoding a mature human growth hormone fused to one of the leader polypeptide variants containing a combination of successively located pro-regions capable of stimulating the secretion of human growth hormone by S yeast cells .cerevisiae. The presence of the KEX2 proteinase recognition site in the fusion region of the target protein and leader polypeptide allows, during the secretion of enzymatic processing, a fusion protein with the participation of KEX2 intracellular proteinase to release mature human somatotropin (containing the corresponding native N-terminal amino acid residue). Expression vectors also contain the region of initiation of replication of the endogenous 2-μm yeast plasmid, which ensures their ability to be maintained in the episomal multi-copy state in S. cerevisiae yeast cells. In order to stabilize the plasmid, the functional gene PGK1 of S. cerevisiae yeast is introduced into the vectors.

Stage 3 - the obtained variants of expression vectors transform the recipient strain of S. cerevisiae yeast. Preferably, the strain S. cerevisiae D721W, which is diploid, is used as the recipient strain of yeast, which provides increased stability of its expression characteristics. The strain S. cerevisiae D721W contains homozygous mutations in the chromosomal alleles of the PGK1 structural gene encoding phosphoglycerate kinase, which ensures stable maintenance of the vector containing the functional PGK1 gene on media containing any single carbon source digested by Saccharomyces cerevisiae yeast, and encoding a protein is a repressor of the GAL1 promoter, which provides galactone-independent expression of genes under the control of the GAL1 promoter.

Stage 4 - based on the results of a comparative analysis of the obtained transformants, a yeast strain is selected that provides the most effective secretion of human growth hormone.

Stage 5 - using the selected yeast strain and fermentation equipment, carry out the biosynthesis of human growth hormone.

The construction of the inventive strain-producer of secreted human growth hormone is carried out as described above using, as a combination of pro-regions, doubled pro-region of S. cerevisiae yeast α-factor. The result is a strain of yeast Saccharomyces cerevisiae SCR-GH-FF - producer of mature human growth hormone, which is deposited in the All-Russian collection of industrial microorganisms (VKPM) as Saccharomyces cerevisiae VKPM Y-3580.

Characterization of the claimed strain

Genotype:

SCR-GH-FF (a / α leu2 / leu2 ura3 / ura3 trp1 / trp1 :: TRP1 pgk1 :: URA3 / pgk1 :: URA3 gal80 :: LEU2 / gal80 :: LEU2 lys7 / LYS7 his3 / HIS3 his4 / HIS4 STA2 / STA2 suc ° / SUC2) / pPDX3-FF-GH

Morphological features:

When cultured at a temperature of 28 ° C for 48 hours on an agarized YPD medium of the following composition (in wt.%): Peptone 2, yeast extract 1, glucose 2, agar 2, the rest of the water, the cells of the claimed strain are oval, 3-7 microns in diameter. Cells are budded. The budding is true, many-sided. True mycelium is not formed.

Colonies are as follows:

1) on an agarized YPD medium, colonies of white color with a smooth edge, a matte surface, a lenticular profile and a creamy consistency;

2) on an agar medium with starch (composition in wt.%: Peptone 2, yeast extract 1, starch 1, agar 2, rest water) white colonies with a patterned edge, a matte surface, a lenticular profile and a crumbly consistency.

Growth in a liquid medium with starch: at 28 ° C during the first 24 hours of cultivation, the liquid is cloudy, the precipitate is white, does not clump, does not form wall films.

Physico-chemical characteristics: Optional anaerobic. The growth temperature is 20-33 ° C (optimum 28 ° C). The pH of the cultivation of 3.8-7.4 (optimum - 5.0).

Assimilation of carbon sources: Ferments glucose, fructose, maltose, sucrose, dextrins, starch. Does not ferment lactose, galactose, inulin, xylose, arabinose.

Assimilation of nitrogen sources: Absorbs amino acids, ammonium sulfate, ammonium nitrate.

Pathogenicity: The inventive strain of Saccharomyces cerevisiae VKPM Y-3580 is non-pathogenic.

Storage: The strain is stored at a temperature of -70 ° C in a 20% aqueous solution of glycerol. Storage on an agarized rich medium with glucose is possible for 3 months at + 4 ° C.

Stability: Stable for 20 consecutive transfers on agarized YPD medium at a temperature of 28 ° C.

Growth hormone production: Secrets mature human growth hormone in an amount of 1-2 g / l.

The claimed group of inventions is illustrated by the following figure, in which is an electrophoregram of samples of culture fluid obtained by culturing strains producing human growth hormone (example 9): SCR-GH-F (d.1), SCR-GH-150 (d.2) , SCR-GH-150F (door 3), SCR-GH-FF (door 4), SCR-GH-150x2 (door 5) and SCR-GH-FFF (door 5 and 6). The tracks contain the total proteins of the samples obtained from 10 μl of culture fluid. An electrophoregram shows a sample of a pharmaceutical drug for human somatotropin (Rastan, 2.5 μg, d.K), as well as molecular weight markers (d.M, the molecular weight of protein markers (in kDa) on the left. Measurement data are shown in the lower part of the electrophoregram the content of human growth hormone (mg / l) in the corresponding samples of the culture fluid obtained using enzyme-linked immunosorbent assay (ELISA).

Example 1. Construction of DNA encoding a leader polypeptide containing the sequence of the doubled pro-region of the S. factor yeast α-factor S. cerevisiae

The construction of a DNA sequence encoding a leader polypeptide comprising the protein sequence of the doubled pro-region of S. cerevisiae yeast α-factor is carried out using plasmid p91-1 containing an expression cassette comprising the GAL1 promoter and the mature human growth hormone gene fused in a single reading frame with the DNA sequence encoding the leader pre-pro-region of the S. factor cerevisiae α-factor [RU2009145487]. Plasmid p91-1 was digested at the NcoI and XhoI sites. The largest of the formed DNA fragments, comprising the vector part of the plasmid and the DNA sequence encoding the pro-region of the yeast α-factor fused to the sequence of the promoter region of the yeast GAL1 gene, is eluted from the gel and is called fragment-1. Then, using PCR primers N449 (5′-atatagatctccagtcaacactacaa) and a standard reverse pUC primer called N169 (5′-gagcggataacaatttcacacagg), the p91-1 plasmid DNA fragment amplifying the sequence of the pro-region of the α-factor yeast and c Mature human growth hormone gene. The amplified DNA fragment is cleaved at the unique terminal sites of BglII and XhoI and is called fragment-2. Then, as a result of annealing one another of two synthetic oligonucleotides N573 (5′-catgttggaattcg) and N574 (5′-gatccgaattccaa), a synthetic adapter called fragment-3 is obtained. As a result of co-ligation of the three obtained DNA fragments, plasmid pUC18x-GAL1pFF-GH is obtained.

The resulting plasmid pUC18x-GAL1pFF-GH contains the mature human growth hormone gene fused in one reading frame with a DNA fragment encoding a leader polypeptide consisting of a signal peptide and a sequence of a doubled pro-region of the yeast α-factor.

Example 2. Construction of DNA encoding a leader polypeptide containing the sequence of triple the pro-region of the α-factor of the yeast factor S. cerevisiae

The construction of a DNA sequence encoding a leader polypeptide comprising the protein sequence of the triple pro-region of S. cerevisiae yeast α-factor is carried out using plasmid pUC18x-GAL1pFF-GH (Example 1). For this, the plasmid pUC18x-GAL1pFF-GH is cleaved at the NcoI and XhoI sites. The largest of the resulting DNA fragments, comprising the vector part of the plasmid and the sequence of the promoter region of the yeast GAL1 gene, fused to the leader polypeptide containing the doubled pro-region of the yeast α-factor, is called fragment-4. As a result of co-ligation of three DNA fragments, fragments 2 and 3 (Example 1) and fragment-4, plasmid pUC18x-GAL1pFFF-GH is obtained.

The resulting plasmid pUC18x-GAL1pFFF-GH contains the mature human growth hormone gene fused in the same reading frame as a DNA fragment encoding a leader polypeptide consisting of a signal peptide and a sequence of triple yeast α-factor pro-region.

Example 3. Construction of DNA encoding a leader polypeptide containing the sequence of the doubled pro-region of the S. cerevisiae yeast HSP150 protein

The construction of the leader polypeptide gene containing the doubled pro-region of S. cerevisiae yeast HSP150 protein is carried out using plasmid p91-15 containing the expression cassette, including the GAL1 promoter and the mature human growth hormone gene, fused in the same reading frame with the DNA sequence encoding the leader polypeptide containing the sequence of the pro-region (H) of the S. cerevisiae yeast HSP150 protein [RU 2009145487]. Plasmid p91-15 is cleaved at the NcoI and XhoI sites. The largest of the resulting DNA fragments, comprising the vector part of the plasmid and the DNA sequence encoding the HSP150 protein pro-region fused to the yeast GAL1 promoter region, is eluted from the gel and is called fragment-5. Next, using N616 primers (5′-aatagatctgcctatgctccatctga) and a standard reverse pUC primer called N169 (5′-gagcggataacaatttcacacagg) for PCR, the DNA fragment of the plasmid p91-15, encoding the sequence of the S. cerevisia cerevisia oblast protein region HSP1, is amplified and a gene of mature human growth hormone fused to it. The amplified DNA fragment is cleaved at the unique terminal sites of BglII and XhoI and is called fragment 6. As a result of the joint ligation of three DNA fragments, fragment 3 (example 1) and fragments 5 and 6, plasmid pUC18x-GAL1matHH-GH is obtained.

The resulting plasmid pUC18x-GAL1matHH-GH contains a mature human growth hormone gene fused to a DNA fragment encoding a leader polypeptide including the sequence of the doubled pro-region of S.cerevisiae yeast protein HSP150.

Example 4. Construction of DNA encoding a leader polypeptide containing the sequence of the pro-region of the S. cerevisiae yeast HSP150 protein and the pro-region of the S. cerevisiae yeast α-factor

Using PCR primers N449 (5′-atatagatctccagtcaacactacaa) and a standard reverse pUC primer called N169 (5′-gagcggataacaatttcacacagg), amplify the DNA fragment of plasmid p91-1 [EN 2009145487], which contains the DNA sequence that encodes the α-region-factor encoding factor S. cerevisiae yeast and the mature human growth hormone gene fused to it. The amplified DNA fragment is cleaved at the unique terminal sites of BglII and XhoI and is called fragment 7.

As a result of the joint ligation of three DNA fragments, fragment 5 (example 3), fragment 3 (example 1) and fragment 7, the plasmid pUC18x-GALlmatHF-GH is obtained.

The obtained plasmid pUC18x-GAL1matHF-GH contains a mature human growth hormone gene fused to a DNA fragment encoding a leader polypeptide comprising the sequences of the pro-region of S. cerevisiae yeast protein HSP150 and the S. cerevisiae yeast α-pro-region.

Example 5. Construction of variants of yeast expression vectors, including various combinations of pro-regions

Construction is carried out using a laboratory bireplicon vector pPDX3. The pPDX3 vector contains the region of replication initiation of the endogenous 2-μm yeast plasmid, which ensures its ability to be maintained in the yeast Saccharomyces cerevisiae cells in an episomal multi-copy state, the vector also carries a functional allele of the yeast PGK1 gene, which ensures its selective maintenance in yeast cells mutated by the chromosomal allele of this gene. The pPDX3 vector contains a single substitution in the NcoI site sequence (ccatgg changed to ccatga), localized in the region of the URA3 structural gene, which does not lead to inactivation of this gene.

The design is as follows. The pPDX3 vector DNA is digested with HindIII / XhoI and ligated with the HindIII / XhoI DNA fragment of plasmid p91-1, or pUC18x-GAL1pFF-GH, or pUC18x-GAL1pFFF-GH, or pUC-15, or pUC18x-GAL1matHH-GH, or pUC18x GAL1matHF-GH, comprising the sequences of the GAL1 promoter and the mature human growth hormone gene fused to a DNA sequence encoding the corresponding leader polypeptide variant. The result is expression vectors pPDX3-F-GH, or pPDX3-FF-GH, or pPDX3-FFF-GH, or pPDX3-H-GH, or pPDX3-HH-GH, or pPDX3-HF-GH, respectively.

The obtained birepliconic expression vectors contain a mature human growth hormone gene fused to DNA sequences encoding variants of leader polypeptides containing single (F), doubled (FF) or tripled (FFF) pro-regions of yeast α-factor, single (H) doubled (HH) pro-region of S. cerevisiae yeast HSP150 protein or a combination of S. cerevisiae yeast HSP150 pro-region and S. cerevisiae yeast α-pro-region (HF), respectively.

Example 6. Construction of a recipient strain of yeast S.cerevisiae D721W

The recipient strain of S. cerevisiae D721W is a derivative of the laboratory strain of S. cerevisiae D721 / pPDX3. Cells of strain D721 / pPDX3 carry homozygous mutations in the chromosomal alleles of the structural genes PGK1 and GAL80.

Strain D721W is constructed in two stages. At the first stage, the cells of the laboratory strain D721 / pPDX3 are transformed with a DNA fragment obtained by any method containing the native TRce gene of S. cerevisiae yeast. The transformation is carried out according to the method of [Ito H, Fukuda Y, Murata K, Kimura A; J Bacteriol. 1983; 153: 163-168]. Transformants are selected on standard mineral medium for growing tryptophan-free yeast.

Select one of the obtained transformants and call it a strain of S. cerevisiae D721W / pPDX3.

At the second stage, cells of strain D721W / pPDX3 are freed from the episomal vector pPDX3 located in them. To do this, they are cultivated on YPGE agar medium of the following composition in wt.%: Bactopeptone 2, yeast extract 1, bactoagar 2, ethanol 2, glycerin 3, and the rest is water. On this medium, cells carrying the pPDX3 plasmid, which contains the PGK1 gene, and cells that have lost them grow equally efficiently. Non-plasmid clones are selected for lack of growth on YPD medium with glucose. The resulting plasmid-free strain is called S. cerevisiae D721W strain.

Strain D721W is used as recipient for transformation with yeast bireplikonny expression vectors.

Example 7. Obtaining the inventive strain and other strains of yeast S. cerevisiae-producers of secreted human growth hormone

The yeast strain SCR-GH-F, or SCR-GH-FF, or SCR-GH-FFF, or SCR-GH-H, or SCR-GH-HH, or SCR-GH-HF is obtained by transformation of the cells of the recipient strain D721W expression vectors pPDX3-F-GH, or pPDX3-FF-GH, or pPDX3-FFF-GH, pPDX3-H-GH, or pPDX3-HH-GH, or pPDX3-HF-GH, respectively. To carry out the transformation, cells of strain D721W were cultured for 18-24 hours at a temperature of 28 ° C on YPGE agar medium. The transformation of the grown cells is carried out according to the method of Ito [Ito et al., J Bacteriol. 1983; 153: 163-168]. Transformants are selected for their ability to grow on YPD medium.

To analyze the level of growth hormone secretion, the cells of the obtained transformants were cultured on YPD medium for 44 hours at 28 ° C on a rotary shaker at a speed of 250 rpm. Next, analyze the level of secreted growth hormone in the culture medium of yeast. As a negative control, strain D721W / pPDX2 is used.

The results of the analysis (Figure 1) show that the strains SCR-GH-FF, SCR-GH-FFF, SCR-GH-HH and SCR-GH-HF, the growth hormone secretion of which is directed by leader polypeptides comprising a combination of pro- regions, SCR-GH-F and SCR-GH-H strains are significantly superior in productivity, in which growth hormone secretion is directed by the pre-pro-region of yeast α-factor or the pre-pro-region of HSP150 protein, respectively. At the same time, the level of growth hormone secretion by strains SCR-GH-FF, SCR-GH-FFF, SCR-GH-HH and SCR-GH-HF is at least 200 mg / l, which is higher than that of strains SCR-H1 and SCR-H2 [RU 2009145487].

The strain SCR-GH-FF is deposited in the All-Russian collection of industrial microorganisms (VKPM) as a strain of Saccharomyces cerevisiae VKPM Y-3580.

Example 8. Microbiological synthesis of human growth hormone using a strain VKPM Y-3580

To obtain seed, the VKPM strain Y-3580 was grown in YPD medium on a shaker (250 rpm) at a temperature of 28 ° C for 24 hours.

50 ml of seed is used to seed 3 L of Anglicon fermenter containing 950 ml of YPD medium. Fermentation is carried out at a temperature of 28 ° C, aeration of 1 l / 1 l / min and a stirring speed of 1000 rpm. 24 hours after sowing the fermenter begin feeding with an aqueous solution of the following composition (in grams per liter): peptone 50, yeast extract 50, sucrose 200; at a rate of 8 ml / h, and establish the pH-stating of the culture at a pH of 6.8, using solutions for 10% sulfuric acid and 10% NaOH. The total fermentation time is 72 hours.

The amount of secreted human growth hormone by the VKPM strain Y-3580 under these conditions is 2 g / l.

Claims (2)

1. A method for the microbiological synthesis of secreted human growth hormone by Saccharomyces cerevisiae yeast cells, comprising culturing a producer strain containing a DNA sequence encoding a mature human growth hormone under the control of a promoter that expresses human growth hormone in Saccharomyces cerevisiae yeast cells, while the human is mature with one reading frame with a leader polypeptide comprising a signal peptide and a sequence performing in yeast cells fu a region of the pro-region, characterized in that as a pro-region of the leader polypeptide, a combination of either two or three consecutively located pro-regions of the α factor of the yeast Saccharomyces cerevisiae, or a double pro-region of the protein HSP150 of the yeast Saccharomyces cerevisiae, or a combination of the pro-region protein HSP150 of the yeast Saccharomyces cerevisiae and the pro-region of the α-factor of the yeast Saccharomyces cerevisiae. 2. The yeast strain Saccharomyces cerevisiae VKPM Y-3580 is a producer of secreted human growth hormone, designed to implement a variant of the method according to claim 1, comprising the use of two consecutively located pro-regions of the α-factor of the yeast Saccharomyces cerevisiae as a pro-region of the leader polypeptide obtained on based on the recipient diploid strain of Saccharomyces cerevisiae containing homozygous mutations of the PGK1 gene and GAL80 gene, by transforming the recipient strain with an expression vector containing the PGK1 gene and under control it GAL1 promoter DNA sequence encoding mature human growth hormone signal peptide of yeast α-factor of Saccharomyces cerevisiae, and a combination of two successive pro region of yeast α-factor of Saccharomyces cerevisiae.

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