The strain of the yeast saccharomyces cerevisiae 1-60-d (msil), the producer of recombinant interleukin-2 and method thereof

 

The invention relates to the field of genetic engineering and biotechnology and can be used in microbiological and medical industries. By crossing strains 33-D (MATpheA10 ade2-144,717 his7-1 lys9-A21 ura3-52 trp1-289 leu2-3,112) and 5-D (Mata arg6 pho3) receive the strain 60-D, which then transform obtained from the plasmid pADP1 linear fragment of the gene RER, inactivated by insertions ADE2 gene. The use of the proposed strain of the yeast Saccharomyces cerevisiae 1-60-D provides a high level of production of interleukin-2. 2 S. p. f-crystals, 1 Il.

The invention relates to biotechnology, genetic engineering, Microbiology and medical industry and is a yeast strain producing interleukin-2 human rights and how to obtain it.

Interleukin-2 belongs to the family of cytokines, signaling molecules of the immune system that are involved in the formation and regulation of protective reactions of an organism. Within the immune system cytokines realize the relationship between non-specific defensive reactions and specific immunity. At the level of the organism cytokines provide a link between the immune, nervous, endocrine species for genetic engineering and biotechnology, many cytokines are obtained in the form of recombinant drugs, some of them are used in clinical practice in the form of medicines (cetlinski S. A. and other Endogenous immunomodulators. SPb., 1992). The use of recombinant cytokines fills a deficit of endogenous molecules, enhances the efficacy of immunotherapy due to adequate and targeted drug correction of immune dysfunction. Currently, immunotherapy with recombinant cytokines is one of the most promising and ever-expanding areas of immunopharmacology.

The main function of the interleukin-2 is to provide the cellular component of adaptive immunity. Interleukin-2 is a factor of growth and differentiation of T-lymphocytes, NK-cells, provides differentiation of T-killers and contributes to the manifestation of the functional activity of T-helper cells. Interleukin-2 enhances the synthesis of immunoglobulins pre-activated B-lymphocytes. It directly affects monocytes expressing the receptor of interleukin-2. A wide spectrum of biological activities of interleukin-2 makes it a key component of the immune system (Taniguchi T. and Minani Y., Cell, 1993, v.73, p.5-8; Jeal W. and K. L. Goa, BioDrugs, 1997, v.7, p.285-317; Rodriquez Z. M. et al. Infection, 1996, v.24, p.115-120; Yarylo A. A. OS is two is the use of microorganisms as producers of these proteins. The use of non-pathogenic microorganisms (yeast) that do not contain toxic and pyrogenic factors, as producers of human proteins allows the use of recombinant proteins in clinical practice. Previously created in the laboratory of biochemical genetics, Binii SPBU strain of the yeast Saccharomyces cerevisiae VKPM Y-791 producing interleukin-2 was synthesized 30-50 mg of biologically active recombinant protein per liter of culture medium (Myasnikov A. N. and other Patent SU # 1770359, the priority of 24 March 1988). Dosage form of recombinant yeast interleukin-2 human Roncoleukin successfully used for treatment of septic conditions, the number of oncological and infectious diseases (Roncoleukinrecombinant interleukin-2. Edited by Kozlov C. K. SPb., 2001). The prospect of widespread use of recombinant yeast interleukin-2 in clinical practice aims at increasing the level of production of this protein in yeast cells.

The task, which directed the present invention is the creation of a strain of the yeast Saccharomyces cerevisiae producer of recombinant interleukin-2 and how it is received.

For Rebeca and developed a method of obtaining a strain of the yeast Saccharomyces cerevisiae - the producer of recombinant interleukin-2.

Strain 1-60-D get from strain 60-D, which transform the plasmid pADPI to disrupt gene RER. To obtain strain 60-D hybrid strains 33-D (MATpheA10 ade2-144,717 his7-l lys9-A21 ura3-52 trp1-289 leu2-3,112) and 5-D (MATa arg6 pho3) Peterhof genetic collection (Andrianova C. M., Inge-Vechtomov, S. G. Directory (index) of the Peterhof genetic collection of yeast Saccharomyces cerevisiae. L., 1988). In spore progeny selected strain 60-D containing the ade2 mutation-144,717, leu2-3,112, lys9-A21, arg6, pho3.

For targeted gene disorders RER in strain 60-D construct plasmid pADP1.

The drawing shows the circuit construction of plasmid pADP1.

At the first stage synthesize gene RER by PCR, using as positive primer sequence 5'-ggaattcatgttcagcttga-3', containing the restriction site for EcoRI, and as a negative primer sequence 5'-tcccccgggtttagctcaaa-3', containing the restriction site for SmaI, as a matrix using the chromosomal DNA of Saccharomyces cerevisiae.

The synthesized gene RER (1,23 etc., O.) was isolated by electrophoresis in a 0.7% agarose gel, treated with restrictase EcoRI and SmaI and are ligated with plasmid pUC19 (2,7, etc., O.) (Roberts R. J. Nucl. Acids Res., the shape of cells of strain DH5Escherichia coli (F'/endAl hsdR17 (r-km+k) supE44 thi-1 recAl gyrA (Nalr) relAl(lacZYA-argF)U169 deoR (80dlac(lacZ)M15), using restriction analysis of selected transformants containing the plasmid pUC19-PEP4 (3,93, etc., O.). To prove the identity of the gene RER synthesized using a PCR amplified gene RER Saccharomyces cerevisiae spend the determination of its nucleotide sequence (Sanger F. S. et al. Proc. Natl. Acad. Sci., 1977, v.74, p.5463-5467).

Plasmid pUC19-PEP4 treated with restriction enzyme EcoRV and are ligated with a DNA fragment containing the ADE2 gene. This fragment obtained by restriction analysis of plasmid pTL1 (Ostanin, K. C. and other Biopolymers and cell, 1988, T. 4, S. 272-279) restriction enzyme Pst1, separation of DNA fragments by electrophoresis in a 0.7% agarose gel, marquee size 2,80, etc., of O., blunting sticky ends using DNA polymerase I of phage T4. After ligation mixture transformed cells of strain DH5Escherichia coli (F'/endAl hsdR17 (r-km+k) supE44 thi-1 recAl gyrA (Nalr) relAl(lacZYA-argF)U169 deoR (80dlac(lacZ)M15) and using restriction analysis of selected transformants, sod is gel isolated fragment 4,33, etc., of O., containing PEP4::ADE2, which is used for transformation of strain 60-D. Transformants are selected on selective medium containing adenine. Violation of gene RER check to reduce the activity repressible alkaline phosphatase on the environment, containing no inorganic phosphate (PEP) (Hemmings B. A. et al., Proc. Natl. Acad. Sci., 1981, v.78, p.435-439). The fact of integration PEP4::ADE2 in chromosome prove by means of PCR using the above primers RER and chromosomal DNA of the transformants as template.

To obtain strain-producer of interleukin-2 human strain 1-60-D injected plasmid pJDB(MSIL) (Myasnikov A. N., Smirnov, M. N., Avot A. Ya. and other Russian Patent SU 1770359, BI No. 39, 1992) and select transformants on medium without adenine and leucine.

The gene expression of interleukin-2 men in the plasmid pJDB(MSIL) is under the control of a yeast promoter RNA containing region, providing activation of transcription in the absence of inorganic phosphate in the culture medium, as well as the region of transcription initiation. The promoter of the gene RNA is among the most strong yeast promoters. The expression level of genes under the control RNA promoter, effectively regulated by exogenous inorganic phosphate. Plasmid p stable and vysokoopasnykh vectors yeast. It contains LEU2 gene of yeast, which is a selective marker for selection of transformants.

Strain-recipient 1-60-D carrying a mutation in the gene LEU2 that allows you to select transformants carrying plasmid pJDB(MSIL). In addition, it contains a disrupted gene RER. Mutation in this gene leads to the lack of activity of proteases a and b and carboxypeptidase Y in yeast cells, which is accompanied by increased stability of heterologous recombinant proteins (Hisch H. H. et al. In: Walton E. F., Yarranton, G. T., Eds., Molecular and Cell Biology of Yeast, 1989, p.134-200). In contrast to strain the prototype VKPM Y-791, strain 1-60-D not bear mutations in the gene RNA. Mutation in this gene is accompanied by constitutive expression of interleukin-2 person, regardless of the concentration of inorganic phosphate. Synthesis of heterologous protein during the whole period of cultivation of the producer strain is a metabolic load for the yeast cells and often leads to mitotic instability of recombinant plasmids (A. Miyanohara et al., Proc. Natl. Acad. Sci., 1983, v.80, p.1-5).

The strain of the yeast Saccharomyces cerevisiae 1-60-D (MSIL) is characterized by the following features.

Morphological features.

The cells are round, slightly oval in shape, with a size of 5-10 microns, part of the cells is on the surface of hanicheskih environments:

PEP - 2% peptone, 2% glucose,

PEPTO - 2% peptone, 2% glucose, 0.1% of one-deputizing potassium phosphate,

YEPD - 2% peptone, 2% glucose, 1% yeast extract.

In addition, the cells grow well on mineral environment SC: of 0.67% Yeast Nitrogen Base (“Difco, USA), 2% glucose, 30 mg/l lysine, 30 mg/l of arginine, as well as other synthetic environments for yeast containing 30 mg/l lysine, 30 mg/l arginine.

During growth on solid media the cells form a smooth, round colonies with a matte surface, light cream color, smooth edge.

With the growth in liquid media intensive form a smooth suspension. Culture has a characteristic odor of yeast.

Physiological and biochemical characteristics.

Cells grow in the range from 4 to 37. The optimal temperature for growing is 30. With the growth in aerobic conditions cells significantly zachisliaut environment. The optimum pH for growth is 3.5 to 5.5.

As a carbon source, cells can use many simple compounds, such as glucose, sucrose, glycerin.

As the nitrogen source provided supplements of lysine and arginine cells use mineral salts in the ammonium form, amino acids, urea.

Cells capable of Aaron is no need in leucine and adenine.

To obtain strain 1-60-D with a damaged genome RER use plasmid pADP1.

An EXAMPLE of the METHOD

At the first stage synthesize gene RER by PCR, using as template the chromosomal DNA of Saccharomyces cerevisiae (see drawing). 0.1 μg of the chromosomal DNA, dissolved in 5 μl of THE buffer, add 1 ál of 0.5 M NaOH and heated at 85for 3 minutes and Then the sample was quickly transferred to ice, add 1 ál of 0.5 M Hcl and then used in PCR.

As a positive primer used the sequence 5'-ggaattcatgttcagcttga-3', containing the restriction site for EcoRI, and as a negative primer sequence 5'-tcccccgggtttagctcaaa-3', containing the restriction site for SmaI. The sample for PCR contains 5 ál of the matrix 30 PM of each primer (2 μl), 10 μl of 10-fold solution deoxynucleotidase (dNTP) containing 1.25 mm of each dNTP (dATP, dTTP, DSTF, dCTP), 10 ál of 10x buffer for PCR (100 mM KCl, 100 TM (NH4)2SO4, 200 TM Tris-HCl, pH 8.8, 20 mM MgSO4, 1% Triton X-100). In a sample is added to distilled H2O to a final volume of 100 μl.

Next, the sample is heated 5 min at 95With, cool, add 2.5% (2.5 ál) Vent DNA polymerase (“BioLabs”) and �="https://img.russianpatents.com/chr/176.gif">(annealing of primers), 1 min at 72(polymerase reaction). After PCR, the sample is incubated at 725 minutes

For evidence that is synthesized during the PCR gene RER, conduct electrophoresis in a 0.7% agarose gel in TBE buffer (0.1 M Tris-borate buffer, pH 8.3, containing 1 mm EDTA). The reaction mixture contribute to the wells of the agarose gel and carry out the separation of DNA fragments within 1-2 hours after the separating cut a strip of the gel containing the DNA fragment size of 1.23, etc., of O., the corresponding gene RER Saccharomyces cerevisiae. DNA isolation from agarose gel performed according to the method developed by QIAGEN. A strip of the gel with a DNA fragment is placed in a test tube and add a solution QX1 (300 μl per 100 mg of gel). The sample is heated to 50add the reagent QIAEX (10 μl of 5 μg DNA) and incubated at 50for 10 minutes, stirring occasionally. Further centrifuged for 30 s at 15,000 rpm, the supernatant discarded, the residue is extracted twice with solutions QX2 and QX3, remove the supernatant by centrifugation at 15,000 rpm for 30 C. the Residue is dried in air, dissolved in 20 μl of THE buffer, centrifuged for 30 s at 15,000 rpm, the supernatant is transferred into a coRI and SmaI carried out in 10 mm Tris-chloride buffer (pH 7.5), containing 50 mm sodium chloride, 10 mm magnesium chloride and 1 mm dithiothreitol. To 5 μg of plasmid DNA in a volume of 20 ál add 5 units of each restrictase, after which the sample is incubated for 25 h at 37°. Next, perform a ligation with plasmid pUC19 previously processed by the same restrictases.

Plasmid pUC19 (2,7, etc., O.) was isolated from the bacterial cells of Escherichia coli grown overnight in 1 l of LB nutrient medium (1% peptone, 0.5% of yeast extract, 1% NaCl) containing ampicillin at a concentration of 50 mg/l of Cells harvested by centrifugation at 5000 rpm for 10 min at 4, suspended in 20 ml of 25 mm Tris-chloride buffer (pH 8.0) containing 10 mm EDTA and 50 mm glucose, add 30 mg of lysozyme and incubated for 10 min at room temperature. Next, add 40 ml of 0.2 M sodium hydroxide containing 1% sodium dodecyl sulfate, gently mixed and incubated for 10 min at 4. The solution is neutralized by adding 30 ml of 3 M sodium acetate (rn,0) and incubated for 10 min at 4. Then centrifuged at 14000 rpm for 40 min at 4. To the supernatant add 0.6 volume of isopropanol �//img.russianpatents.com/chr/176.gif">. The precipitate is washed with 70% ethanol, dried in vacuum and dissolved in 4 ml of distilled water. Next add to 4.2 g of cesium chloride and 0.36 ml solution of ethidium bromide (10 mg/ml). The resulting solution was incubated for 1 h at 4, then centrifuged at 15,000 rpm for 15 min. the Supernatant centrifuged at 70000 rpm for 16 h in the TL100 centrifuge (Beckman). After centrifugation select band of plasmid DNA (the lower of the two fluorescent under ultraviolet light stripes), twice extracted with ethidium bromide equal volume of isoamyl alcohol, diluted twice with distilled water and precipitated plasmid DNA with two volumes of ethyl alcohol and 1/15 volume of 3 M sodium acetate (pH 5.0). The precipitate is collected by centrifugation at 10,000 rpm for 10 min, washed with 70% ethanol and dissolved in 0.5-1 ml of buffer TE (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 means of electrophoresis in a 0.7% agarose gel in TBE buffer.

Hydrolysis of plasmid pUC19 by restrictase EcoRI and SmaI carried out under the conditions described for the cleavage of the synthetic is the method of firm QIAGEN, excluding stage heating.

To obtain the plasmid pUC19-PEP4 spend ligation of plasmids pUC19, hydrolyzed by restrictase EcoRI and SmaI, and EcoRI/SmaI fragment of the gene PEP 4 Saccharomyces cerevisiae synthesized using PCR. To do this, mix 0.5 μg DNA vector and 0.1 μg of the DNA insertion in 10 μl of 70 mm Tris-chloride buffer (pH of 7.6) containing 5 mm dithiothreitol, 5 mm magnesium chloride, 1 mm ATP, add 10 units of DNA ligase of phage T4 and incubated at 14throughout the night.

Received ligase mixture of transforming cells of strain DH5Escherichia coli (F’/endAl hsdR17 (r-km+k) supE44 thi-1 recAl gyrA (Nalr) relAl(lacZYA-argF)U169 deoR (80dlac(lacZ)M15). For this purpose, cells of Escherichia coli grown in 100 ml LB medium at 37until the culture density of cell suspension corresponding to 0.4-0.6 units of optical density at a wavelength of 550 nm. The cell suspension is cooled in an ice bath, centrifuged at 5000 rpm for 10 min at 4. Cells spenderat in 100 ml of 10 mm sodium chloride, collected by centrifugation in the same conditions. The cells are suspended in 50 ml of 75 mm the cult chloride in 1 ml of 75 mm calcium chloride. To a suspension of competent cells add glycerol to a final concentration of 15%, divided into aliquots and stored at -70. Before the transformation of a suspension of competent cells thawed in an ice bath, add ligase mixture and incubated in ice bath for 40 minutes the cells are subjected to the action of heat shock at 42within 2 min, and then incubated in 1.5 ml of LB medium at 37within 1 h, the Cells are harvested by centrifugation at 5000 rpm for 10 min and plated on Petri dishes with LB medium containing 2% agar and 50 mg/l ampicillin. Cup incubated at 37within 12-16 hours

From the higher individual clones of transformants produce plasmid DNA pUC19-PEP4 (3,93, etc., O.) using the methodology used to obtain the plasmid pUC19, except that cells of Escherichia coli grown in 10 ml LB, and, respectively, the volumes of all solutions is reduced 100 times. In addition, instead of the stage centrifugation in density gradient cesium chloride spend processing DNA pancreatic RNase. For this purpose, nucleic acid, precipitated with isopropyl alcohol, dissolved in 100 μl of THE buffer, add 10 ál of the solution P is sledding to cleavage of the synthesized using PCR gene RER Saccharomyces cerevisiae, linearized plasmid DNA secrete by the method described above company QIAGEN, excluding stage of heating, and are ligated with a DNA fragment containing the ADE2 gene of Saccharomyces cerevisiae. This fragment obtained by restriction analysis of plasmid pTL1 the restriction enzyme Pst1. The selection plasmids pTL1 and its hydrolysis by restriction enzyme Pst1 carried out under the conditions described above for plasmid pUC19. After hydrolysis plasmids pTL1 the restriction enzyme Pst1 carry out the separation of DNA fragments by electrophoresis in a 0.7% agarose gel and isolated fragment of 2.8, etc., acting by the method described above company QIAGEN.

The resulting fragment is treated with DNA polymerase I of phage T4 in 50 mm Tris-chloride buffer (pH of 7.8) containing 10 mm magnesium chloride, 10 mm dithiothreitol, 1 mm ATP, 25 μg/ml bovine serum albumin. To 5 µg DNA in a volume of 20 ál add 5 units of DNA polymerase I of phage T4, after which the sample is incubated for 8 h at 16C. and are ligated with plasmid pUC19-PEP4 treated with restriction enzyme EcoRV. Ligase mixture of transforming cells of strain DH5Escherichia coli (F'/endAl hsdR17 (r-km+k) supE44 thi-1 recAl gyrA (Nalr) relAl(lacZYA-argF)U169 deoR (80dlac(lacZ)M15, and using restricciones PvuII and by agarose gel electrophoresis isolated fragment 4,33, etc., of O., containing the gene RER with a built-in ADE2 gene.

The linear fragment of plasmid pADP1 size 4,33 T. p. O. transform cells of strain 60-D to obtain strain 1-60-D with a damaged genome RER.

To obtain strain 60-D hybrid strains 33-D (MATpheAl0 ade2-144,717 his7-1 lys9-A21 IgA-52 trpl-289 leu2-3,112) and 5-D (Mata arg6 pho3) Peterhof genetic collection. Received diploid grows on mineral medium without added amino acids. Diploid transferred to a medium with sodium acetate (1% sodium acetate, 0.5% of potassium chloride) and 4-5 days sporelings culture used for isolation of haploid spores. Sporelings culture treated with medical ether for 5 min and plated on complete medium YEPD. Grown on YEPD medium disputes subcultured in full Wednesday and after 2 days method prints transferred to selective medium, determine the phenotype of segregants and take away the strain 60-D containing the ade2 mutation-144,717, leu2-3,112, lys9-A21, arg6, pho3.

For targeted gene disorders RER strain 60-D transform the linear fragment of plasmid pADP1 size 4,33, etc., of O., which contains the gene RER with built-ADE2 gene. Cells of yeast strain 60-D grown in 100 ml of YEPD medium until the culture optical is the donkey which is suspended in 0.3 ml of 100 mm solution of lithium acetate and incubated at 30within 30 minutes To 50 μl of the resulting suspension of cells add 0.1-1 μg of plasmid DNA, 50 μg DNA, salmon sperm, previously denatured by heating for 10 min at 100), and 0.3 ml of 100 mm lithium acetate containing 40% polyethylene glycol 4000. Next, the sample is incubated for 30 min at 30and 20 min at 42placed at 15 C in an ice bath and centrifuged for 10 s at 10000 rpm Cells suspended in 1 ml of sterile water and plated on solid medium SC containing containing 30 mg/l lysine, 30 mg/l of arginine, 60 mg/l leucine. Transformants are selected based on their ability to grow on medium that does not contain adenine. Clones of transformants grow within 4-6 days.

Violation of gene RER check to reduce the activity repressible alkaline phosphatase on the environment, PEP. The fact of integration PEP4::ADE2 in chromosome prove, by means of PCR using the primers RER. As a matrix using the chromosomal DNA of the transformants.

Then, the resulting strain 1-60-D with a damaged genome RER create producing strains of interleukin-2.

To obtain a strain of the yeast Saccharomyces cerevisiae 1-60-D (MSIL) - producer of interleukin-2 human, yeast cells of the strain 1-60-D transform and, relevant 2-4 units absorption at a wavelength of 600 nm. Cells are washed twice with sterile water, and then suspended in 0.3 ml of 100 mm solution of lithium acetate and incubated at 30within 30 minutes To 50 μl of the resulting suspension of cells add 0.1-1 μg of plasmid DNA, 50 μg DNA, salmon sperm, previously denatured by heating for 10 min at 100), and 0.3 ml of 100 mm lithium acetate containing 40% polyethylene glycol 4000. Next, the sample is incubated for 30 min at 30and 20 min at 42placed at 15 C in an ice bath and centrifuged for 10 s at 10000 rpm Cells suspended in 1 ml of sterile water and plated on solid medium SC containing 30 mg/l lysine, 30 mg/l arginine. Clones of transformants grow within 4-6 days.

To analyze the production of interleukin-2 human cell transformants are grown in 50 ml liquid medium PEP to the stationary phase of growth. Cells are harvested by centrifugation at 3000 rpm for 10 min, washed with water, suspended in 1 ml of 50 mm Tris-chloride buffer (pH 7.5) containing 1 mm PMSF (phenylmethylsulfonyl) add 1 g of glass beads (0.5 mm) and destroy in the cage “Braun” within 1 minutes Provera and suspended in 1 ml of 50 mm nutrifaster buffer (pH 7.0), containing 2% sodium dodecyl sulfate and 5% 2-mercaptoethanol, and incubated for 5 min in a boiling water bath. After incubation the sample was centrifuged 15 min at 12,000 rpm and the supernatant determine the content of interleukin-2 by means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent hybridization with specific antibodies to interleukin-2. Separation of proteins is carried out in a 15% polyacrylamide gel in the standard system buffers (electrode buffer: 25 mm Tris, 192 mm glycine, 0.1% sodium dodecyl sulphate, pH 8.3; buffer for gel: 375 mm Tris-chloride buffer, pH 8.8). In parallel to carry out the separation of proteins of a control strain 1-60-D grown and exposed to identical conditions. As molecular weight standards used carbonic anhydrase (31,0 kDa), trypsin inhibitor (21.5 in kDa), myoglobin (of 16.9 kDa), lysozyme (14,4 kDa). After electrophoresis proteins centurybut, soaking the gel for 15 min in 10 mm Tris-chloride buffer (pH 7.5) containing 4M urea, 20 mm EDTA, and transferred to nitrocellulose membrane in 25 mm Tris-192 mm glycine buffer (pH 8.3) containing 20% methanol, at 30-40, for 1.5 hours Then the membrane is incubated in TBST buffer (10 mm -/img.russianpatents.com/chr/176.gif">. Then place the membrane in the same buffer containing diluted 1000 times murine monoclonal antibodies to interleukin-2 (Boehringer) and incubated 2 h at 37. Then thrice washed membrane with TBST buffer and incubated 1 h at 37diluted to 7,000 times the conjugate of species-specific antibodies to mouse IgG and horseradish peroxidase (Protein contour, St. Petersburg). After washing the membrane with PBST buffer (58 mm disubstituted phosphate, 17 mm one-deputizing phosphate, 68 mm NaCl, 0.1% tween-20) add the substrate solution for peroxidase: 0,02% D(3,'3-diaminobenzidine tetrahydrochloride), 0,006% hydrogen peroxide in 10 mm Tris-chloride buffer, pH 7.5. Simultaneously stained gels of 0.15% solution of Kumasi G250 in 25% isopropanol and 10% acetic acid and washed in 10% acetic acid. When comparing the spectrum of proteins in strain 1-60-D (MSIL) detect the appearance of an additional protein band, which gives a positive reaction with antibodies to interleukin-2 person, with a molecular mass of approximately 15.4 kDa, which corresponds to the molecular mass of interleukin-2. The level of synthesis of interleukin-2 determined by comparing the intensity of staining bands of recombinantly 1-60-D (MSIL) synthesize about 60-65 mg of interleukin-2 per liter of yeast culture.

Recombinant interleukin-2 synthesized in yeast cells, has biological activity, providing the proliferation of interleukin-2-dependent T-cell line mouse CTLL-2 (Gearing, A., Thorpe, R. J. Immunol. Meth., 1988, v.114, p.3-9). The activity of recombinant interleukin-2 is not less than 10 million units/L.

In sum, we can conclude that the advantage of the established strain of the yeast Saccharomyces cerevisiae 1-60-D (MSIL) in comparison with the prototype VKPM Y-791 is a higher level of production of interleukin-2 and greater stability of the recombinant protein, which improves the yield of the target product.

The strain of the yeast Saccharomyces cerevisiae 1-60-D (MSIL) producing interleukin-2 deposited in Russian national collection of industrial microorganisms under the number VKPM Y-3079.

Claims

1. The strain of the yeast Saccharomyces cerevisiae 1-60-D (MSIL), deposited in the all-Union collection of industrial microorganisms under the number VKPM Y-3079 - producer of recombinant interleukin-2.

2. A method of obtaining a strain of the yeast Saccharomyces cerevisiae 1-60-D (MSIL), which consists in the fact that the strain 33-D (MATpheA10 ade2-144,717 his7-1 lys9-A21 ura3-52 trp1-289 leu2-3,112) crossed with shtam the config segregant impose a linear fragment of the gene RER, inactivated by insertions ADE2 gene, which is derived from plasmid D1, and selected transformant 1-60-D growing on selective medium without adenine, in the selected transformant 1-60-D injected plasmid JDB (MSIL), containing the promoter and terminator of transcription of the gene RNA yeast, the coding part of the gene interleukin-2 for synthesis of recombinant interleukin-2 human yeast cells in the absence of inorganic phosphate in the culture medium.

 

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