RU2203950C1 - Strain of yeast pichia pastoris as producer of leukocyte human interferon-16, recombinant plasmid dna phin and method for its construction - Google Patents

Abstract

FIELD: genetic engineering. SUBSTANCE: invention reports the construction of recombinant plasmid DNA pHIN representing Eco RI-Xho I-fragment of bifunctional bacterial-yeast vector pPIC19 ligated with encoding region of human gene alpha-interferon-16 but with deletion in region encoding signaling peptide. As result of transformation of the strain Pichia pastoris PS 99 with plasmid pHIN the recombinant strain Pichia pastoris PS 104 (pHIN) is obtained that provides both effective synthesis of human alpha-interferon- 16 (15 mg per 1 l of culture) and secretion of the end product by cells. Invention can be used in microbiological and medicinal industry. EFFECT: valuable properties of strain, increased yield of interferon. 3 cl, 2 dwg

Description

 The invention relates to biotechnology, genetic engineering, the microbiological and medical industries, and is a yeast strain producing human leukocyte interferon-16, which contains a plasmid constructed in vitro that provides the synthesis and secretion of human leukocyte interferon-16.

 Human leukocyte interferon, alpha-interferon-16, belongs to an extensive group of evolutionarily related proteins, called interferons. At present, 23 species of human alpha-interferons that differ in amino acid composition but share common functional characteristics have been isolated and characterized. The formation of interferons is stimulated by the action of viruses on various groups of cells. Interferons are able to activate the antiviral defense mechanisms of the cell. Clinical trials have shown the possibility of using alpha-interferons for the treatment of a number of viral diseases, such as hepatitis B, C, D, herpes infection, keratitis, influenza (Guo JT et al. J. Virol., 2000, v.75, p.8516- 8523; Prescrire Int., 2001, v. 10, p. 17-21). In addition, at present, alpha-interferons together with other drugs are actively used in the treatment of various oncological diseases (prostate cancer, renal cell cancer, breast cancer, leukemia, malignant skin diseases) and human autoimmune diseases (Molchanov O.E. and et al. Current trends in the immunotherapy of malignant tumors, 2001; Ershov F.I. The interferon system is normal and pathological, 1996; Kramer G. et al. J. Interferon Cytokine Res., 2001, v.21, p. 475-484). The species-specificity of interferons does not allow the use of alpha-interferons from other mammals in clinical trials. The use of donor blood or a leukocyte culture as a source of alpha interferons does not produce the required amounts of the drug, and in addition, it carries the risk of infection with hepatitis B, C and HIV.

 A promising approach for obtaining human alpha-interferon-16 in significant quantities is the use of microorganisms as producers of this drug. There are a number of strains of Escherichia coli, Pseudomorias putida, Sacharomyces cerevisiae producing various types of human alpha interferons. The most common are producers of recombinant alpha-interferon-2, obtained on the basis of prokarytic microorganisms. But Escherichiacoli and Pseudomonas putida are conditionally pathogenic for humans and alpha-interferon preparations obtained from bacterial cells may contain endotoxins. The complete release of alpha-interferon from an impurity of endotoxins, which is a prerequisite for its use in clinical practice, greatly complicates the procedure for purification of recombinant protein. Currently, the domestic preparation of recombinant human alpha-interferon-2b, synthesized by Pseudomonas putida cells and manufactured by Vector-Medica CJSC, is registered and approved for use. However, prolonged use of it can lead to undesirable side effects due to the conditional pathogenicity of the producer strain (Ershov F.I. The interferon system is normal and pathological, 1996).

 All of the above indicates the promise of creating producer strains of human alpha-interferon-16 based on other microorganisms, in particular yeast. 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. The yeast strains Saccharomyces cerevisiae, producers of human leukocyte interferon-16, which were previously created in the laboratory of biochemical genetics at the Research Institute of St. Petersburg State University, were characterized by low productivity. The production level ranged from 0.1 mg to 1 mg of biologically active alpha-interferon-16 per liter of culture (Myasnikov A.N. et al. SU 1573577, 1988; Myasnikov A.N. et al. SU 1584188, 1988). In addition, the carbohydrate component of proteins excreted by saccharomycete yeast differs significantly from that of higher eukaryotes glycoproteins (Kukuruzinska M. A. et al. Ann. Rev. Biochem., 1987, v. 56, p. 915-944). The carbohydrate portion of glycoproteins is a strong antigenic determinant; therefore, recombinant human alpha interferon-16 secreted by Saccharomyces cerevisiae yeast can only be used for laboratory research (Ballou C.E. J. Biol. Chem, 1970, v.245, p.1197-1203).

 The use of methylotrophic yeast Pichia pastoris for heterologous expression is of particular interest. This, firstly, makes it possible to significantly increase the yield of recombinant proteins. Secondly, glycoproteins secreted by Pichia pastoris yeast do not contain mannose residues, linked by α1-3 bonds and are strong antigenic determinants, which makes it possible to obtain, along with intracellular, authentic secretory forms of recombinant proteins (Montesino R. et al. Protein Expr. Purif. , 1998, v.l4, p. 197-207).

 Obtaining a secretory producer of human alpha-interferon-16 is especially important. Protein is a glycoprotein and only in the process of secretion does it undergo glycosylation and acquire the correct conformation. The accumulation of recombinant protein in the culture fluid greatly simplifies the procedure for its purification.

 The problem to which the proposed objects of the invention are directed is to create a strain of Pichia pastoris yeast that synthesizes human alpha interferon-16 and secrete it into the culture fluid, create recombinant pHIN plasmid DNA that provides the synthesis and secretion of human leukocyte interferon-16, and develop a method its design.

 To solve this problem, a method was developed for constructing pHIN recombinant plasmid DNA, which ensures the synthesis and secretion of leukocyte interferon, as a result, a yeast strain Pichia pastoris is created that synthesizes human alpha-interferon-16 and secrets it into the culture fluid.

The pHIN plasmid DNA, which provides the synthesis and secretion of human alpha-interferon-16 by the yeast cells transformed by it, has a total size of 8.51 kb, (5.6 molecular weight Mda), consists of the following elements:
- EcoRl-Xhol - a fragment of plasmid DNA of a bifunctional bacterial yeast vector pIC9 of size 8.00 kbp, including the bacterial gene for resistance to ampicillin, the bacterial region of replication initiation, HIS4 gene of yeast; a fragment of the 5'-non-coding region of the yeast gene AOX1 with a size of 0.95 kbp containing a region that activates transcription of this gene in the presence of methanol as a carbon source in the culture medium; a prepreg of the MFα gene of Saccharomyces cerevisiae yeast 0.27 kbp in size, providing for the secretion of interferon beta into the culture medium; a 0.33 kbp AOX1 gene fragment containing a transcription termination region of this gene; a fragment of the 3'-untranslated region of the AOX1 gene of 0.76 kbp .;
- Xhol-EcoRI — 0.51 kbp fragment containing the coding portion of the human alpha interferon-16 gene except for the region encoding the signal peptide;
- Unique recognition sites of the following restriction enzymes: BamH1 - 0.94, etc. about. ; Xho1 - 1.19 kbp .; EcoR1 - 1.71 kb; Sall - 3.70 kb; Sphl - 5.31 kbp

 The scheme of plasmid DNA pHIN with a restriction map is depicted in figure 1.

 The construction scheme of the plasmid pHIN is shown in Fig.2.

 Plasmid pJDB (MS105) (Myasnikov A.N. et al. SU 1584188, 1988) is used as a template for the synthesis of the human alpha-interferon-16 gene by PCR. A 5'-ccgctcgagaaaagatgtgatctgcctcag oligonucleotide containing the Xhol restriction enzyme site is used as a direct primer. As the reverse primer, a 5'-ggaattcctcaatccttccttcttaatcct oligonucleotide that contains an EcoRl restriction enzyme site is used. The synthesized human alpha-interferon-16 gene 0.51 kbp in size isolated by electrophoresis in a 0.7% agarose gel, digested with restriction enzymes Xhol and EcoRl and ligated with plasmid pPIC9 (Invitrogen) pretreated with the same restriction enzymes.

The resulting ligase mixture was introduced into cells of the strain DH5α Escherichia coli (FVendAl hsdR17 (r _k ^- m _k ⁺ ) supE44 thi-1 recAl gyrA (Nal ^r ) relAl Δ (lacZYA-argF) U169 deoR (φ80dlacΔ (lacZ) M15), using restriction analysis, transformants containing the pHIN plasmid are selected. To prove the identity of the alpha-interferon-16 gene synthesized by PCR, the native human alpha-interferon-16 gene is determined for its nucleotide sequence (Sanger FS et al. Proc. Natl. Acad. Sci ., 1977, v. 74, p. 5463-546).

 The choice of plasmid design for the production of alpha-interferon-16 is due to the following reasons. Plasmid pHIN was obtained on the basis of the shuttle bacterial-yeast integrative vector pPIC9. As a result of the transformation by a linearized plasmid and subsequent homologous recombination, the expression cassette is inserted into the Pichia pastoris yeast chromosome, which ensures stable maintenance of the cloned alpha-interferon-16 gene. The plasmid contains the yeast HIS4 gene, which makes it possible to selectively select transformants when yeast strains with mutations in this gene are used as recipients.

 The expression of the human alpha-interferon-16 gene in the pHIN plasmid is controlled by the promoter of the AOX1 gene, which contains regions that activate transcription in the presence of methanol in the culture medium, as well as a transcription initiation region. The AOX1 gene promoter is one of the most powerful yeast promoters. The level of expression of genes under the control of the AOX1 promoter is effectively regulated by carbon sources. Transcription of the AOX1 gene is completely blocked when yeast is grown on a medium with glucose; on a medium with glycerin, only a basal level of gene expression is observed. The use of methanol as the sole carbon source significantly enhances the expression of the AOX1 gene and, therefore, genes controlled by the AOX1 promoter. This allows you to regulate the synthesis of alpha-interferon-16 in yeast cells. Regulated expression of the cloned gene can significantly reduce the metabolic load on the yeast cell. The presence of the Saccharomyces cerevisiae yeast MFα gene preproregion in the plasmid provides for the secretion of the synthesized human alpha-interferon-16 into the culture fluid.

 As producer of alpha-interferon-16 use strain PS104 (pHIN). Strain PS104 (pHIN) was obtained by transforming the strain of yeast PS99 (his4 pp4 :: PH085) with the plasmid pHIN. Strain PS99 carries a mutation in the HIS4 gene, which makes it possible to selectively select transformants carrying the plasmid pHIN. Mutation in the PEP4 gene leads to a lack of activity of proteases A and B, as well as carboxypeptidase Y in yeast cells, which is accompanied by increased stability of heterologous recombinant proteins (Hisch HH et al. In: Walton EF, Yarranton GT, Eds., Molecular and Cell Biology of Yeast 1989, p.134-200).

 The strain of the yeast Pichia pastoris PS104 (pHIN) is characterized by the following features.

Morphological features
The cells are round, slightly oval in shape, 5-10 microns in size, some of the cells on the surface of the kidney or connected to daughter cells.

Cultural signs
Cells grow well on a complete organic medium YEPD - 2% peptone, 1% yeast extract, 2% glucose or 1% glycerol.

 In addition, cells grow well on SC mineral medium: 1.34% Yeast Nitrogen Base (Difco, USA), 2% glucose (1% glycerol, 0.5% methanol), as well as other synthetic yeast media.

 When growing on solid media, cells form smooth, round colonies with a matte surface, light cream in color, the edge is uneven.

 When grown in liquid media, they form an intense, even suspension. The culture has a characteristic smell of methylotrophic yeast.

Physiological and biochemical characteristics
Cells grow in the range from 4 to 37 ^o C. The optimal growth temperature is 30 ^o C. When growing under aerobic conditions, the cells slightly alkalize the medium. The optimum pH for growth is 4.5-6.5.

 Cells can use many simple compounds as a carbon source, such as glucose, glycerin, methanol.

 As a source of nitrogen, cells can use mineral salts in the ammonium form, amino acids, urea.

 Cells are capable of aerobic and anaerobic growth.

 The essential features of the strain are the lack of need for histidine.

 A method of obtaining a plasmid pHIN is illustrated by the following example.

 EXAMPLE 1

Escherichia coli bacteria cells containing the pJDB plasmid (MS105) were grown at 37 ^{° C.} overnight in 1 L of LB growth medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) containing ampicillin at a concentration of 50 mg / l Cells are harvested by centrifugation at 5,000 rpm for 10 min at 4 ^° C, suspended in 20 ml of 25 mM Tris-chloride buffer (pH 8.0) containing 10 mM EDTA and 50 mM glucose, 30 mg lysozyme are added and incubated for 10 min at room temperature. Then add 40 ml of 0.2 M sodium hydroxide containing 1% sodium dodecyl sulfate, mix gently and incubate for 10 min at 4 ^o C. The solution is neutralized by adding 30 ml of 3 M sodium acetate (pH 5.0) and incubated for 10 min at 4 ^o C. After that, centrifuged at 14000 rpm for 40 min at 4 ^o C. To the supernatant add 0.6 volumes of isopropyl alcohol, incubated for 20 minutes at room temperature and centrifuged at 14000 rpm for 20 minutes at 20 ^o C. The resulting precipitate was washed with 70% ethanol, dried in vacuo and a solution of dissolved in 4 ml of distilled water. Next, 4.2 g of cesium chloride and 0.36 ml of ethidium bromide solution (10 mg / ml) are added. The resulting solution was kept 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 TL100 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 isoamyl alcohol, diluted twice with distilled water, and plasmid DNA is precipitated with two volumes of ethyl alcohol and 1/15 volume of 3 M sodium acetate ( pH 5.0). The precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed with 70% ethanol 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).

 The resulting plasmid pJDB (MS105) is used as a template for the synthesis of the human alpha-interferon-16 gene using PCR.

To 0.1 μg of plasmid DNA dissolved in 5 μl of TE buffer, 1 μl of 0.5 M NaOH was added and heated at 85 ^{° C.} for 3 minutes. Then the sample is quickly transferred to ice, 1 μl of 0.5 M Hcl is added and then used in PCR.

 A 5'-ccgctcgagaaaagatgtgatctgcctcag oligonucleotide containing the Xhol restriction enzyme site is used as a direct primer. As the reverse primer, a 5'-ggaattcctcaatccttccttcttaatcct oligonucleotide that contains an EcoRl restriction enzyme site is used.

The PCR sample contains 5 μl of the matrix, 30 rM of each primer (2 μl each), 10 μl of a 10-fold solution of deoxynucleotide triphosphates (dNTP) containing 1.25 mM of each dNTP (dATP, dTTP, dGTP, dCTP), 10 μl 10- multiple PCR buffer (100 mM KCl, 100 tM (NH ₄ ) ₂ SO _4, 200 tM Tris-HCl, pH 8.8, 20 mM MgSO ₄ , 1% Triton X-100). Distilled H ₂ O is added to the sample to a final volume of 100 μl.

Next, the sample is heated for 5 min at 95 ^o C, cooled, add 2.5 units (2.5 μl) Vent-DNA polymerase ("BioLabs") and carry out 50 PCR cycles under the following conditions: 1 min at 95 ^o C (melting of DNA chains), 1 min at 46 ^o C (annealing of primers), 1 min at 72 ^o C (polymerase reaction). After PCR, the sample is incubated at 72 ^° C. for 5 minutes.

To prove that the human alpha-interferon-16 gene is synthesized during PCR, electrophoresis is performed on a 0.7% agarose gel in TBE buffer. The reaction mixture was added to the wells of an agarose gel and DNA fragments were separated for 1-2 hours under the conditions described above. At the end of the separation, a strip of gel containing a 0.51 DNA fragment of DNA is cut out. about. corresponding to the human alpha interferon-16 gene. Isolation of DNA from agarose gel is carried out according to the method developed by QIAGEN, using special kits manufactured by the company. A strip of gel with a DNA fragment is placed in a tube and QX1 solution (300 μl per 100 mg of gel) is added. The sample is heated to 50 ^° C., QIAEX reagent (10 μl per 5 μg DNA) is added and incubated at 50 ^° C. for 10 minutes, stirring occasionally. Then it is centrifuged for 30 s at 15000 rpm, the supernatant is discarded, the precipitate is extracted twice with QX2 and QX3 solutions, and the supernatant is removed by centrifugation at 15000 rpm for 30 s. The precipitate was dried in air, dissolved in 20 μl of TE buffer, centrifuged for 30 s at 15,000 rpm, the supernatant was transferred to a new tube.

Hydrolysis of a DNA fragment containing the synthesized human alpha-interferon-16 gene with Xhol and EcoRI restriction enzymes is 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 μl add 5 units. each restrictase, after which the sample is incubated for 25 hours at 37 ^o C.

 Isolation of plasmid pIC9 is carried out under conditions similar to plasmid pJDB (MS105). The hydrolysis of its restriction enzymes Xhol and EcoRI is carried out under the conditions described for the cleavage of the human alpha-interferon-16 gene synthesized by PCR. Linearized plasmid DNA was isolated by the QIAGEN method described above, excluding the heating step.

To obtain the pHIN plasmid, ligation of plasmid pIPC9 hydrolyzed with restriction enzymes Xhol and EcoRI and XhoI / EcoRI fragment of the human alpha-interferon-16 gene synthesized by PCR is performed. To do this, mix 0.5 μg of DNA vector and 0.1 μg of DNA insertion in 10 μl of 70 mm Tris-chloride buffer (pH 7.6) containing 5 mm dithiothreitol, 5 mm magnesium chloride, 1 mm ATP, add 10 units Phage T4 DNA ligases and incubated at 14 ^° C. overnight.

Cells of strain DH5 α Escherichia coli (F '/ endAl hsdR17 (r _k ^- m _k ⁺ ) supE44 thi-1 recAl gyrA (Nal ^r ) relAl Δ (lacZYA-argF) U169 deoR (φ80dlacΔ (lacZ) M15) are transformed with the obtained ligase mixture Escherichia coli: For this, Escherichia coli cells are grown in 100 ml of LB medium at 37 ^° C. until the culture reaches a cell suspension density corresponding to 0.4-0.6 optical density units at a wavelength of 550 nm. The cell suspension is cooled in an ice bath, centrifuged at 5000 rev / min for 10 min at 4 ^o C. the cells supendiruyut in 100 ml of 10 mM sodium chloride, collected by centrifugation under the same conditions. Next, cells suspendi comfort 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. Prior to transformation, a suspension of competent cells is thawed in an ice bath, a ligase mixture is added and incubated in an ice bath for 40 minutes. The cells are then 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 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. The plates are incubated at 37 ^° C. for 12-16 hours.

Plasmid DNA was isolated from the grown individual clones of transformants using the technique used to obtain plasmid pJDB (MS105), 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 the centrifugation step in the density gradient of cesium chloride, DNA is treated with pancreatic RNKase. 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 min at 37 ^° C.

 Next, hydrolysis of the obtained plasmid DNA with restriction enzymes EcoRI and Xhol is carried out. With restriction of the desired plasmid pHIN and subsequent electrophoresis in 0.7% agarose gel, fragments of 0.51 and 8.00 kb are detected. From the clone thus identified, the pHIN plasmid is preparatively isolated as described for plasmid pJDB (MS105) and digested with BglII restriction enzyme in 50 mM Tris-chloride buffer (pH 7.5) containing 100 mM sodium chloride, 10 mM magnesium chloride. The BglII fragment of plasmid pP1C9, size 5.63 kbp, was isolated according to the QIAGEN method described above, excluding the heating step, and used to transform yeast cells, as described in Example 2.

 EXAMPLE 2

 To obtain the yeast strain Pichia pastoris, a producer of human alpha-interferon-16, yeast cells of strain PS99 are transformed with the plasmid pHIN.

Yeast cells are grown in 100 ml of YEPD medium at 30 ^° C. until the culture reaches an optical density corresponding to 2-4 absorption units at a wavelength of 600 nm. 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 pre-denatured by heating (10 min at 100 ^° C) and 0.3 ml of a solution of 100 mM lithium acetate containing 40% polyethylene glycol 4000 are added. 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 SC solid medium. Clones of transformants grow in 2-3 days. The grown clones are subcultured onto plates with SC medium containing 2% glucose in separate colonies, then reprinted onto MM medium (1.34% Yeast Nitrogen Base (Difco, USA), 0.5% methanol, 2% agar (Difco ", USA) for the selection of transformants that do not grow on a medium with methanol, which indicates the integration of a foreign gene into the AOXI locus (Met- phenotype).

To analyze the production of alpha-interferon-16 cells of transformants of the Met phenotype, they are grown at 30 ^° C in 100 ml of BMGY liquid medium (2% peptone, 1% yeast extract, 1% glycerol, 10 ml 1M potassium phosphate buffer, pH 6, 0, 1.34% Yeast Nitrogen Base (Difco, USA) to the stationary growth phase for 2 days. Cells are collected by centrifugation at 5000 rpm for 10 min, the supernatant is drained and the whole biomass is transferred into 20 ml of BMMY liquid medium (2% peptone, 1% yeast extract, 0.5% methanol, 10 ml of 1M potassium phosphate buffer, pH 6.0, 1.34% Yeast Nitrogen Base (Difco, USA) to induce al expression alpha-interferon-16. Induction is carried out at 30 ^o C for 4 days. At the end of induction, the culture medium is separated from the cell biomass by centrifugation at 5000 rpm for 10 minutes in the culture medium determine the content of alpha-interferon-16 by electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate and subsequent hybridization with specific antibodies to human alpha interferon. 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 the proteins of the control strain grown under identical conditions is carried out. Carbonic anhydrase, trypsin inhibitor, myoglobin, lysozyme are used as molecular weight standards. At the end of electrophoresis, the proteins are renatured, keeping the gels for 15 min in 10 mM Tris-chloride buffer (pH 7.5) containing 4 M urea, 20 mM EDTA, and 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. 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 37 ^° C. Then placed the membrane into the same buffer containing 1000-fold diluted mouse polyclonal antibodies to human alpha-interferon (Protein circuit), and incubated for 2 hours at 37 ^o C. Next, wash the membrane three times with TBST buffer and incubate for 1 hour at 37 ^o C diluted 7000 times conjugate of species-specific antibodies to mouse immunoglobulins of horseradish peroxidase ("Protein circuit "). 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), a solution of peroxidase substrates was added: 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 0.15% Coomassie G250 solution in 25% isopropanol and 10% acetic acid and washed in 10% acetic acid. When comparing the spectrum of proteins of the two strains, strain PS104 (pHIN) shows the appearance of two additional protein bands that give a clear positive reaction with antibodies to human alpha interferon: the main one with a molecular weight of 20 KDa, which corresponds to the molecular weight of glycosylated alpha interferon-16, and minor - 22 KDa. Both proteins are glycoproteins, as indicated by staining with Schiff's reagent. The appearance of a minor fraction of alpha-interferon-16 with a molecular weight of 22 KDa can be explained by different degrees of glycosylation of the protein molecule. The level of synthesis of alpha-interferon-16 is determined by comparing the color intensity of the band of the recombinant protein with the band of standard alpha-interferon.

 According to the data obtained, yeast cells of strain PS104 (pHIN) synthesize and secrete about 15 mg of alpha-interferon-16 per liter of yeast culture.

 Summarizing the above, it can be concluded that the obtained strain of Pichia pastoris PS104 yeast (pHIN) synthesizes and secrets human alpha-interferon-16 in an amount sufficient to purify it on a laboratory scale. As a result of this purification, preparations of alpha-interferon-16 can be obtained, suitable for studying its biological properties and therapeutic value. The advantage of this producer in comparison with the prototypes VKMP Y-789 and VKMP Y-790 is a higher level of production of alpha-interferon-16 and greater stability of the recombinant protein. Just like the VKMP Y-790 strain, the obtained Pichia pastoris PS104 yeast strain (pHIN) provides for the secretion of alpha-interferon-16, which greatly simplifies the procedure for purification of the recombinant protein. Recombinant alpha-interferon-16 synthesized and secreted by strain PS104 (pHIN) is a glycoprotein, like human alpha-interferon-16, and at the same time does not contain additional mannose residues characteristic of proteins secreted by saccharomycetes.

 The yeast strain Pichia pastoris PS104 (pHIN), a secretory producer of human interferon alpha-16, has been deposited in the All-Russian Collection of Industrial Microorganisms under the number BKPM-Y2949.

Claims (3)

 1. The strain of yeast Рichia pastoris PS 104 (pHIN), deposited in the All-Union collection of industrial microorganisms under the number VKPM -Y2949, is a producer of human alpha-interferon-16.  2. Recombinant plasmid DNA rHIN, providing biosynthesis and secretion of human alpha-interferon-16, having a size of 8.51, so on. (molecular weight 5.6 MDa), consisting of the following elements: DNA fragment of a bifunctional bacterial-yeast vector pRIC9 measuring 8.00 bp bounded by restriction sites EcoRI-XhoI and including the bacterial gene for resistance to ampicillin, the bacterial region of initiation of replication, the HIS4 gene of yeast, a fragment of 0.95 bp containing the promoter of the yeast gene AOX1, a fragment of 0.27 bp containing the preproregion of the MFα gene of the yeast Saccharomyces cerevisiae, providing secretion of alpha-interferon-16 into the culture medium, a fragment measuring 0.33 tp containing the region of the transcription termination of the AOX1 gene, a fragment of the 3'-untranslated region of the AOX1 gene of 0.76 bp ; XhoI-EcoRI-fragment size of 0.51 T. p. containing the coding portion of the human alpha-interferon-16 gene, except for the region encoding the signal peptide, and containing unique recognition sites for the following restrictase enzymes: BamHI - 0.94 bp , ХhoI-1.19 t.p. , EcoRI-1.71 t.p. , SalI - 3.70 t.p. , SphI-5.31, bp  3. A method of constructing a recombinant plasmid DNA pHIN, providing synthesis of alpha-interferon-16 in Pichia pastoris yeast cells and its secretion into the culture fluid, which consists in the fact that the human alpha-interferon-16 gene, with the exception of the region encoding the signal peptide, is obtained by using PCR using the pJDB plasmid template (MS105), the direct primer 5'-ccgctcgagaaaagatgtgatctgcctcag containing the restriction enzyme site XhoI, and the reverse primer 5'-ggaattcctcaatccttctctctcttaatcct containing the 16 restriction enzyme enzyme vayut digested with XhoI and EcoRI and ligated with plasmid rRIC9 pretreated with the same restriction enzymes, and then ligation mixture transformed Escherichia coli cells and clones containing the recombinant plasmid DNA rHIN.

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