Method of producing recombinant protein, modified through attachment of human albumin from yeast culture fluid

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.

EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.

3 cl, 1 tbl, 5 ex

 

The invention relates to the field of biotechnology, medicine and pharmacology, relates to a method of production of recombinant proteins having therapeutic activity and modified by the addition of albumin human, including the technology of cultivation of strains producing and subsequent purification of target proteins. The present invention is based in part on the discovery of the fact that the hybrid proteins containing albumin of human blood plasma, attached to the target protein, have improved pharmacokinetic properties, such as a longer half-life, which improves the quality of life of patients (Yeh P et al. Proc. Natl. Acad.Sci. 1992. V.89. P.1904-1908; Smith, B. et al. Bioconjugate Chem. 2001. V.12. P.750-756; Halpem W. et al. Pharm. Res. 2002. V.19. P.1720-1729). The active part of the hybrid protein may represent proteins of different nature, which possess biological activity, allowing their use as medicines. For example, it can be enzymes, enzyme inhibitors, hormones, interferons and interleukins. Receiving modified interferons and interleukins, with a broad range of activities, especially true.

Known methods for producing recombinant proteins, modified albumin human, yeast Kluyveromyces lactis (patent US 6,686,179, published February 3, 2004), yeast Saccaromyces cerevisiae (patent US 7,045,318, published on may 16, 2006), bacteria, yeast Saccharomyces cerevisiae and mammalian cells (US patent 6,946,134, published September 20, 2005; US patent 6,994,857 published 7 February 2006; US patent 7,238,667, published July 3, 2007). Bacteria as prokaryotic organisms are not capable of post-translational modification of proteins characteristic of eukaryotic organisms, but due to the conditional pathogenicity prokaryotic strains - producers of recombinant proteins can contain impurities endotoxins, so prolonged use of recombinant proteins of the bacterial nature accompanied by undesirable side effects (Primrose S.B. J. Appl. Bacteriol. 1986. V.61. P.99-116; Ershov FI System of interferon in norm and pathology, 1996). Expression system, based on the culture of mammalian cells, allow you to get authentic heterologous proteins. A significant drawback of these systems is the complexity of working with cell cultures and the complexity of obtaining transgenic plants and animals, which increases the cost of the resulting recombinant protein.

The lack of expression of yeast .lactis and S.cerevisiae is the use of expression plasmids containing the genes for antibiotic resistance that creates the risk of horizontal transfer. In addition, proteins secreted by these yeasts, hyperslices is profiled, that is accompanied by changes in their physico-chemical properties (Schultz L.D. et al. // Gene. 1987. V.54. RI 13-123; Eckart M.R., Bussineau C.M. // Curr. Opin. Biotechnol. 1996. V.7. P.525-530).

Devoid of these shortcomings, the expression system of the yeast Pichia pastoris. Use as producers of the methylotrophic yeast Pichia pastoris has the potential to greatly increase the yield of recombinant proteins and avoid hyperglycosylated of secreted heterologous proteins (Cereghino J.L., Cregg J.M. FEMS Microbiol.Rev. 2000. V.24. P.45-66). The accumulation of recombinant proteins into the culture fluid greatly simplifies purification. However, the yield of the final product depends largely on the cultivation technology of the producer strain, isolation and purification of the recombinant hybrid protein.

A method of obtaining recombinant proteins, modified albumin human, yeast K. Lactis (patent US 6,686,179, published February 3, 2004), the closest to the claimed method, selected as a prototype. The known method lies in the cultivation at 30°C recombinant strains of yeast in a nutrient medium for growth of cells and the synthesis of the target protein, the cell removal, ultrafiltration and subsequent dialysis culture medium, affinity chromatography sorbents type Blue-Trisacryl, the two-stage ion-exchange chromatography sorbents type S Sepharose FF and the Q Sepharose FF, the dialysis.

The disadvantages of this method are its multi-stage and low technology, which limits the possibility of the use of this scheme for the industrial production of recombinant hybrid proteins.

The technical result of the proposed method is to simplify and increase the efficiency of purification of the target protein at the expense of a new method of cultivating yeast - producers, ensuring the production of the target protein having biological activity comparable to the unmodified protein, which allows to obtain biologically active hybrid proteins suitable for creation on their basis of medicines, and also creates the possibility of industrial production of recombinant hybrid proteins.

This technical result is achieved in that in a method of producing recombinant proteins, modified by the addition of albumin human, from the culture fluid of yeast, including the cultivation of strains of yeast in a nutrient medium for growth of cells and the synthesis of target proteins, cell removal, concentrated environment, the precipitation of the target protein and its purification using gel filtration and affinity chromatography, in accordance with the claimed method is cultivated strains of the yeast Pichia pastoris, which are the two who are the producers of hybrid proteins (albumin-interleukin-2 and albumin-16 alpha-interferon person), the cultivation is carried out in a modified environment, the stage of the induction of synthesis of the target protein is carried out at 20-25°C, the cleaning is performed by precipitation of the target protein at 75% saturation of ammonium sulfate or 25% polyethylene glycol 3350, gel-filtration on the sorbent type Sephacryl HR 200 or BioRad P-300 and affinity chromatography on Cibacron F3GA. This concentration of culture medium is performed with the use of membrane AR-0.3-PS.

In addition, this technical result is achieved by gel-filtration on the sorbent type Sephacryl HR 200 or BioRad P-300 is performed using as an equilibrating and eluting solution of 20 mm sodium acetate buffer, pH 5.5.

In addition, this technical result is achieved by affinity chromatography on Cibacron F3GA are using as a balanced solution of 20 mm sodium acetate buffer, pH 5.5; washing implement consistent use of equilibrating buffer solution and equilibrating buffer solution containing 0.5 M sodium chloride, elution of the target protein is performed with a concentration gradient of potassium thiocyanate 0.5-1.0 M in 20 mm sodium acetate buffer, pH 5.5 or 50 mm Tris-HCl, pH 7.5 to 8.5.

In the proposed method the specified technical result is achieved therefore by using recombinant strain drogi the th Pichia pastoris PS106(pPIC9HAbIL-2) - producer of the hybrid protein albumin-interleukin-2 person, previously obtained by the authors of the present invention (the decision to grant a patent dated 23 April 2007 on the application of EN 2006110098), and strain of the yeast P. pastoris PS106 (pPIC9HAbIFNa-16)obtained by the method claimed earlier by the authors of the present invention (the decision to grant a patent dated 23 April 2007 on the application of EN 2006110098) - producer of the hybrid protein albumin-16 alpha-interferon man, the cultivation of strains in a modified nutrient medium and the induction of the synthesis of the target protein, the cell removal, the concentration of culture medium, the deposition of the target protein by ammonium sulfate or polyethylene glycol (PEG) 3350, gel filtration on sorbents type Sephacryl HR 200 or BioRad P-300, affinity chromatography sorbents type Cibacron F3GA.

The optimal conditions of the individual stages of obtaining the target hybrid proteins are the following:

- cultivation of strains - producers in the modified BMGY medium containing 0.05 M potassium phosphate buffer, pH 6.0;

- induction of the synthesis of the target protein at 22-25°C in a modified environment AMM containing 0.05 M potassium phosphate buffer, pH 6.0;

- concentration of culture medium using membrane AR-0.3-PS;

- the precipitation of the target protein at 75% saturation of ammonium sulfate or 25% polyethylene glycol (PEG) 3350;

- dissolution floor is obtained precipitate, containing the target hybrid protein in 20 mm sodium acetate buffer, pH 5.5;

- conducting gel filtration on sorbents type Sephacryl HR 200 or BioRad P-300 in 20 mm sodium acetate buffer, pH 5.5;

- carrying out affinity chromatography sorbents type Cibacron F3GA using as a balanced solution of 20 mm sodium acetate buffer, pH 5.5; removing the impurity proteins 0.5 M sodium chloride in equilibrating buffer, the elution of the target protein by the concentration gradient of potassium thiocyanate 0.5-1.0 M in 20 mm sodium acetate buffer, pH 5.5 or 50 mm Tris-HCl, pH 7.5 to 8.5.

Significant differences of the proposed method from the prototype are:

- use as producers of hybrid proteins of the yeast P. pastoris PS106(pPIC9HAbIL-2) - producer of the hybrid protein albumin-interleukin-2 and PS106(pPIC9HAbIFNa-16) - producer of the hybrid protein albumin-16 alpha-interferon person;

- cultivation of yeast strains in the modified BMGY medium containing 0.05 M potassium phosphate buffer, pH 6.0;

- conducting phase induction of the synthesis of the target protein at 20-25°C in a modified environment AMM containing 0.05 M potassium phosphate buffer, pH 6.0, which allows to obtain higher yield of hybrid proteins;

- consistent use of the stages of ultrafiltration and precipitation of the target protein by ammonium sulfate or polie what ilerliyorum (PEG) 3350, leading to a considerable reduction of the volume of culture medium in the first stages of cleaning, which is important when scaling the manufacturing process of the target proteins;

- two-step chromatographic purification of hybrid proteins on the sorbent type Sephacryl HR 200 or BioRad P-300 and Cibacron F3GA, allowing to obtain biologically active hybrid proteins.

The nature and advantages of the claimed invention is illustrated by the following examples.

Example 1. Cultivation of strains of yeast P. pastoris producing hybrid proteins albumin-interleukin-2 and albumin-16 alpha-interferon. Then take 1 ml of the suspension of the yeast P. pastoris PS106 (pPIC9HAbIL-2) - producer of the hybrid protein albumin-interleukin-2 or PS106 (pPIC9HAbIFNa-16) -producer of the hybrid protein albumin-16 alpha-interferon person stored in 15% glycerol at -70°C, were made in 50 ml of modified BMGY medium (table 1), in comparison with the standard environment, the concentration of potassium phosphate buffer 2 times less, and cultivated at 30°C on a rocking chair with stirring (200-220 rpm) for 24-36 hours before reaching an optical density D600from 2.5 to 4.0, preferably of 3.0. Then 25 ml of the obtained supernatant is transferred into 1 liter of modified environment and cultured in BMGY 1.6 liter fermenter Bioflo III (New Brunwick Sci. Co.) at 30°C with stirring (30-450 rpm) for 68-72 hours before full utilization of glycerol. Then sterile collect cells by centrifugation at 6-7 Tyson/min for 15 minutes at 4°C and transferred into a 1 l modified environment AMM, which unlike BMGY medium as the sole carbon source contains methanol. Adding methanol is the induction of the synthesis of the target protein. The concentration of methanol is maintained in the range from 0.5% to 2.0%, preferably from 0.75 to 1.0%. Cultivation on the induction stage is carried out at 20-25°C, preferably at 22°C for 96-120 hours, preferably 100-110 hours. The production of the target protein are tested using electrophoresis of proteins cultural liquid in reducing conditions. The decrease in the concentration of inorganic phosphate by using a modified environment enhances the production of hybrid proteins.

Table 1
Composition of modified nutrient medium BMGY
Component environmentQuantity for l
MgSO4×7H2O0.5 g
CaCl20.4 g
(NH4)2SO43.0 g
yeast extract10.0 g
peptone20,0 g
Glycerin10 ml
Potassium-phosphate buffer 1 M, pH 6.050 ml
Biotin1.8 mg
CuSO4×5H2O16,6 mg
Nal0.35 mg
MnSO411,7 mg
NaMoO4×2H2O0,74 mg
H3IN30.1 mg
CoCl2of 1.18 mg
ZnSO4×7H2O0.2 mg
FeSO4×7H2O0.3 mg

Analysis of proteins secreted by strains-producers, showed that lowering the temperature on the induction stage to 20-25°C prevents proteolytic cleavage of target proteins and thereby increase their output.

Example 2. Purification of the hybrid protein albumin-16 alpha-interferon man. At the end of the process of culturing yeast cells were removed by centrifugation at 6-7 Tyson/min for 15 minutes at +4°C. All subsequent operations were carried out at a temperature of +4°C. the Supernatant was passed through a nitrocellulose filter with a pore diameter of 0.45 μm (Millpore Inc.).

Thus obtained filtrate was concentrated by ultrafiltration installation UPL-0,6 using membrane AR-0.3-PS (CDD "Biotest", Kirishi), cutting compounds with a molecular mass of less than 50 kDa, at this stage was the removal of low molecular weight components of the environment and reduction of the original volume of 100-200 ml Then there was salt precipitation of the target protein, leading to the saturation of ammonium sulfate to 75%. The residue of the target protein was collected by centrifugation at 12-14 Tyson/min for 20 minutes. The precipitate was dissolved in 20 mm sodium acetate buffer, pH 5.5, and was applied to a column containing 280 ml BioRad P-300, which was equilibrated with the same buffer. The first 75 ml was discarded, the fractions containing the target protein were pooled. At this stage, was the liberation from the main mass of impurities.

Thus obtained eluate was applied to a column containing 2.5 ml of affinity sorbent Cibacron F3GA, balanced 20 mm sodium acetate buffer, pH 5.5. Washed the column sequentially with 10 ml equilibrating buffer and 10 ml ravnovesie the surrounding buffer, containing 0.5 M sodium chloride. The elution was carried out with the concentration gradient of potassium thiocyanate from 0.5 M to 1.0 M in 20 mm sodium acetate buffer, pH 5.5 or 50 mm Tris-HCl, pH 7.5 to 8.5. At this stage remove residual impurity proteins that were products of proteolysis albumin, part of the hybrid protein.

Electrophoresis in reducing conditions showed that the use of this purification has led to the preparation of the hybrid protein albumin-16 alpha-interferon person, the impurity content of not more than 5%. The molecular weight of the hybrid protein albumin-16 alpha-interferon person was approximately 86 kDa. The average output from a liter of culture medium was 7-9 mg

Example 3. Purification of the hybrid protein albumin-interleukin-2 people. At the end of the process of culturing yeast cells were removed by centrifugation at 6-7 Tyson/min for 15 minutes at +4°C. All subsequent operations were carried out at a temperature of +4°C. the Supernatant was passed through a nitrocellulose filter with a pore diameter of 0.45 μm (Millpore Inc.).

Thus obtained filtrate was concentrated by ultrafiltration installation UPL-0,6 using membrane AR-0.3-PS (CDD "Biotest", Kirishi), cutting compounds with a molecular mass of less than 50 kDa, at this stage was the removal of low molecular weight comp the components of the environment and reduction of the original volume of 100-200 ml Then besieged the target protein PEG 3350 at 25% saturation. The residue of the target protein was collected by centrifugation at 12-14 Tyson/min for 20 minutes. The precipitate was dissolved in 20 mm sodium acetate buffer, pH 5.5, and was applied to a column containing 200 ml of Sephacryl HR 200, equilibrated with the same buffer. The first 60 ml was discarded, the fractions containing the target protein were pooled. At this stage, was the liberation from the main mass of impurities.

Thus obtained eluate was applied to a column containing 2.5 ml of affinity sorbent Cibacron F3GA, balanced 20 mm sodium acetate buffer, pH 5.5. Washed the column sequentially with 10 ml equilibrating buffer and 10 ml of equilibrating buffer containing 0.5 M sodium chloride. The elution was carried out with the concentration gradient of potassium thiocyanate from 0.5 M to 1.0 M in 20 mm sodium acetate buffer, pH 5.5 or 50 mm Tris-HCl, pH 7.5 to 8.5. At this stage remove residual impurity proteins that were products of proteolysis albumin, part of the hybrid protein.

Electrophoresis in reducing conditions showed that the use of this purification has led to the preparation of the hybrid protein albumin-interleukin-2 person, the impurity content of not more than 5%. The molecular weight of the hybrid protein albumin-interleukin-2 was approximately 82,0 kDa. Average the exit liter of culture medium was 6-7 mg

Example 4. Study of biological activity of the hybrid protein albumin-16 alpha-interferon person.

Biological activity of the purified hybrid protein was tested for its ability to inhibit the cytopathic activity of vesicular stomatitis virus in primary cultures of L-41 skin and muscle tissue of human embryos that are sensitive to interferon alpha-type (P.T. Liu et al. // Protein Expres. Purif. 2001. V.22. R-387). As the standard used human leukocyte interferon CCA 42-28-90-87 (GISC, Moscow).

Cell culture L-41 skin and muscle tissue of the human embryos were grown in a nutrient medium Needle MEM containing 2 mm L-glutamine, 10% serum fruits cows and antibiotics (penicillin and streptomycin, 100 u/ml) (36,0±1,0)°C. After formation of the monolayer of cells were removed from the glass with a mixture of Versene and trypsin in a 1:1 ratio (FS 42-65 g-93; FS 42-3321-96), a mixture of Versene and trypsin was decanted, the cells suspended in a nutrient medium Needle MEM containing L-glutamine and antibiotics (penicillin and streptomycin, 100 u/ml), but without serum fruits cows at a concentration of 150-300×103/ml 200 ál of the prepared suspensions of cells were made in 96-well plates and incubated at (36,0±1,0°C in the atmosphere (5,0±0,5)% CO2within 48-72 hours prior to the formation of a complete monolayer. Remove culture medium was made in the wells at 100 μl Seri is twofold dilutions of the test drug and standard and incubated at (36,0±1,0°C in the atmosphere (5,0±0,5)% CO 2within 24 hours. Then to each well with the test drug and the standard was introduced, the dose of vesicular stomatitis virus strain "Indiana" (deposited in the collection of risk, per No. 11/82)corresponding to 100 TCD50/0.1 ml, and incubated at (36,0±1,0°C in the atmosphere (5,0±0,5)% CO2within 24 hours. Antiviral activity of the test drug was calculated by the formula:

Ax=(ASTD/aSTD)×ax,

where Ax- antiviral activity of the test drug in IU/ml;

AndSTD- antiviral activity of standard IU/ml;

axthe titer of the test drug (reciprocal dilution);

andSTD- title of the standard (the reciprocal of the dilution).

The specific activity of the purified preparation of the hybrid protein albumin-16 alpha-interferon person amounted to 0.4 to 1.7×107IU/mg. Given that the molecular mass of 16 alpha-interferon man - 20 kDa, and the molecular mass of albumin 66 kDa, fraction of active component - 16 alpha-interferon person in the hybrid protein molecule is approximately 23%. On this basis, we can conclude that the attachment of the albumin does not lead to a significant reduction of biological activity of 16 alpha-interferon person, which is 0.3 to 1.0×108IU/mg (RU 2203950, published on 10 may 2003).

Example 5. Investigated the e biological activity of the hybrid protein albumin-interleukin-2 people.

Biological activity of the purified hybrid protein was tested for its ability to provide the proliferation of interleukin-2-dependent T-cell line mouse CTLL-2 (A.J.H. Gearing, Thorpe R. // J. Immunol. Meth. 1988. V.I 14. P.3-9). As the standard used BRMP reference reagent human IL-2 (NCI, USA).

T-lymphocytes mouse line CTLL-2 suspended in a nutrient medium RPMI 1640 containing 2 mm L-glutamine and antibiotics (penicillin and streptomycin, 100 u/ml), 10% serum fruits cows, at a concentration of 1×106/ml and added into 96-well plates, in which, before it was made 100 μl of a series of twofold dilutions of the test drug and standard. The plates were incubated at (36,0±1,0°C in the atmosphere (5,0±0,5)% CO2within 42 hours. Then to each well was brought to 10 μl of 0.005% solution of thiazoline blue (MTT) in RPMI 1640 without supplements and incubated 6 hours at (36,0±1,0°C in the atmosphere (5,0±0,5)% CO2. In viable cells stimulated by interleukin-2 as part of a hybrid protein formed insoluble in aqueous solutions of dark blue crystals formazan. The plates were centrifuged for 5 minutes at 2000 rpm, was removed from the hole supernatant, add 100-200 ál of dimethyl sulfoxide, was shaken until complete dissolution of the crystals formosana and measured the absorption at wavelengths of 540 nm and 690 nm. The activity of the test drug is determined and the mapping of the values of absorption of the test drug and standard.

The specific activity of the purified preparation of the hybrid protein albumin-interleukin-2 was 1.4-to 2.2×105IU/mg. Given that the molecular mass of 0-glycosylated recombinant interleukin-2 person is 16.0 kDa (Vita N. et al. Lymphokine Res. 1990. V.9. Page 67-79), and the molecular mass of albumin 66 kDa, fraction of active component - 16 alpha-interferon man - in hybrid protein molecule is about 20%. On this basis, we can conclude that the attachment of the albumin does not reduce the biological activity of 16 alpha-interferon person, which is 1.0×106IU/mg (RU 2140283, published on 27 October 1999).

Thus, the inventive method of production of recombinant proteins, modified by the addition of albumin human, including the technology of cultivation of strains producing and subsequent purification of target proteins, allows to obtain the target proteins having biological activity comparable to the unmodified protein. Taking into account the fact that the hybrid proteins containing albumin person attached to the target protein, have improved pharmacokinetic properties, we can conclude that on the basis of the purified hybrid proteins: albumin-interleukin-2 and albumin-16 alpha-interferon can be obtained effective pre is Arata for the treatment of various viral diseases (Guo J.T. et al. J. Virol. 2001. V.75. R-8523; Popovich A.M. Interleukin-2: Immunobiology and immunotherapy. 2004; Siebeck N. et al. Am. J. Vet. Res. 2006. V.67. P.1406-1411; Human Genome Sciences Reports. 2007), cancer (E.C. Borden Oncologist. 1998. V.3. P.198-203; Bukowski R. Semin. Oncol. 2000. V.27. P.204-212; Molchanov PU and other Modern trends in immunotherapy of malignant tumors. 2001) and autoimmune diseases (Piskova J. et al. Invest. Ophtalmol. Vis. Sci. 2006. V.47. P.3946-3950).

The claimed method compared to global counterparts is more than a simple allocation of the target protein and higher efficiency by preventing proteolysis of target proteins, which allows to obtain biologically active hybrid proteins suitable for creation on their basis of drugs with improved pharmacokinetic properties for the treatment of diseases of various etiologies, as well as the possibility of obtaining the target proteins on an industrial scale.

1. A method of obtaining a recombinant protein, modified by the addition of albumin human, from the culture fluid of yeast, including the cultivation of strains of yeast in a nutrient medium for growth of cells and the synthesis of the target protein, cell removal, concentration environment, the precipitation of the target protein and its purification using gel filtration and affinity chromatography, characterized in that the cultivated strain of yeast ichia pastoris PS106/pPIC9HAbIL-2 - the producer of the hybrid protein albumin-interleukin-2 person or strain of the yeast Pichia pastoris PS106/pPIC9HAbIFNa-16 producing a hybrid protein albumin-16 alpha-interferon person in a modified environment BMGY with reduced 2 times the concentration of potassium phosphate buffer stage of the induction of synthesis of the target protein is carried out at 20-25°C, albumin-interleukin-2 human purified by precipitation of 25% polyethylene glycol 3350 and distinguish it from the gel-filtration on the sorbent type Sephacryl HR 200, and albumin-16 alpha-interferon human purified by precipitation with 75% saturation with ammonium sulfate and distinguish it from the gel-filtration on the sorbent type BioRad P-300, in conclusion, spend affinity chromatography of the target protein on Cibacron F3GA.

2. The method according to claim 1, characterized in that the gel-filtration on the sorbent type Sephacryl HR 200 or BioRad P-300 is performed using as an equilibrating and eluting solution of 20 mm sodium acetate buffer, pH 5.5.

3. The method according to claim 1, wherein the affinity chromatography on Cibacron F3GA are using as a balanced solution of 20 mm sodium acetate buffer, pH 5.5, while the wash is carried out consistent use of balanced buffer solution and equilibrating buffer solution containing 0.5 M sodium chloride, and the elution of the target protein is performed by gradient concentric and potassium thiocyanate 0.5-1.0 M in 20 mm sodium acetate buffer, pH of 5.5 or 50 mm Tris-HCl, pH 7.5 to 8.5.



 

Same patents:

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

FIELD: medicine; microbiology.

SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.

EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.

3 cl, 3 dwg, 3 ex

FIELD: genetic engineering.

SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.

EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.

9 cl, 6 ex

FIELD: medicine.

SUBSTANCE: peptide is obtained from phage peptide library including set of static peptides with length of 12 aminoacid residues, by affine selection of phage clones containing peptide capable of specific linking to antibodies of benzo[α]pyrene and benzo[α]anthracene. Peptide displays specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene and features molecular weight of 1.3 kDa and registered aminoacid sequence LHLPHHDGVGWG encoded by nucleotide sequence SEQ ID NO:1.

EFFECT: application in medicine as a base for peptide medicine development for immunologic prevention of malignant tumours of humans.

4 dwg, 3 ex

FIELD: biology.

SUBSTANCE: present invention relates to genetic engineering, more specifically to obtaining anticoagulative protein extracted from nematodes (NAP) and can be used in medicine. To obtain a medicinal preparation based on NAP, methanotrophic yeast host cells are cultured, encoding rNAPc2 or rNAPc2/praline, until attaining the desired cell density. NAP is then extracted from the said yeast host cells through cation-exchange chromatography on an expanding layer. To purify the NAP medicinal preparation, hydrophobic-interaction chromatography is used. NAP is extracted and purified at pH levels below 4.

EFFECT: simple and more efficient method of obtaining anticoagulation proteins from nematodes.

25 cl, 8 dwg, 7 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: claimed are expression vectors for obtaining IL-21 in E.coli cells. IL-21 coding nucleotide sequence, included in composition of novel vectors, contains modifications aimed at optimisation of codons and secondary mRNA structure for translation in E.coli. As a result of transformation with claimed vector structures E.coli strains suitable for industrial scale application have been obtained. Methods of wide scale IL-21 production which use said strains have been elaborated, allowing to obtain more than 1g/l of recombinant cytokine.

EFFECT: novel compounds possess useful biological properties.

14 cl, 1 dwg, 12 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.

EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.

6 cl, 11 dwg, 9 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to production of hormones and can be used for culturing invertebrates. Gonadotrophin, selected from the invertebrate Asterina pectinifera is a peptide with molecular weight 4500-4900, it has two subunits, the protein structure of which is combined with SS-bridges, formed between residues of SH cysteine contained in the subunits.

EFFECT: interfusion and oxidation of these two subunits after synthesis allow producing gonadotrophin having gonadal promoting activity.

4 cl, 8 dwg, 1 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.

60 cl, 16 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.

EFFECT: improved pharmacokinetic properties of erythropoietin.

23 cl, 14 ex, 6 tbl, 11 dwg

Fused proteins il-7 // 2369616

FIELD: medicine.

SUBSTANCE: there is provided fused protein, including immunoglobulin chain and the molecule IL-7 or its fragment, displaying activity typical to IL-7, that is modified in comparison with IL-7 of wild type, where modification in IL-7 represents amino-acid residues in positions 70 and 91, that are glycated, and amino-acid residue in the position 116 is nonglycated. There is proposed pharmaceutical composition, containing described protein. There is provided the method fro production of described fused protein, including: host cell transformation by DNA, coding specified fused protein IL-7, host cell culture and collection of fused protein IL-7.

EFFECT: invention can be applied for treatment of disorders, accompanied by immune deficiencies.

27 cl, 16 dwg, 1 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: according to the invention there is provided protein fused with albumine comprising two or more tandem-oriented polypeptides GLP-1 or its fragments containing (7-36 A8G)), nucleic acid molecules coding albumine proteins according to the investigation as well as vectors containing the specified nucleic acids, host cells transformed by vectors comprising the specified nucleic acids, methods of albumine protein preparation according to the invention and their application methods. Moreover, the present invention refers to pharmaceutical composition containing fused proteins of albumine, treatment of diseases, disorders and conditions using fused proteins of albumine according to the invention.

EFFECT: improved therapeutic polypeptide stability.

34 cl, 81 ex, 11 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.

EFFECT: enlarging the application range of medicine.

9 dwg

FIELD: medicine.

SUBSTANCE: there is offered method for identification and/or verification of inhibitors of receptor tyrosine kinases that involves application of a new test system which represents a yeast host cell containing an expression vector including a nucleic acid sequence that encodes fused protein essentially consisting of a complete cytoplasmic part of analysed receptor tyrosine kinase and the dimerisation domain and, if necessary, in addition including anchoring sequence for fused protein in a membrane wherein expression of fused protein conduces to termination of cell proliferation. The method provides production of specified host cells being in contact with a candidate compound and identification of inhibitors of tested tyrosine kinase activity as a result of cultivation on the assumption that inhibition of tyrosine kinase activity with a candidate compound causes restoration of proliferation process.

EFFECT: prospected application of the invention is related to development of selective therapeutic, including anticancer, agents.

13 cl, 5 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.

EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.

22 cl, 13 dwg, 8 tbl

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biotechnology and can be used in biomedicine for producing hyaluronan. Proposal is given of a method of producing hyaluronan, involving culturing Bacillus host cells in conditions, suitable for obtaining hyaluronan, and extraction of the target product from the culture medium. The Bacillus host cell contains a genetic structure, comprising a promoter, functionally active in the given cell, and encoding a region, consisting of a nucleotide sequence, encoding streptococcal hyaluronansynthase (hasA); sequence encoding UDP-glucose-6-dehydrogenase Bacillus (tuaD) or a similar enzyme of streptococcal origin (hasB), and a sequence, encoding bacterial or streptococcal UDP-glucose pyrophosphorylase (gtaB and hasC respectively). Use of the invented method provides for production of considerable quantities of hyaluronan with good examined, nonpathogenic cellular system.

EFFECT: obtaining considerable of hyaluronan with good examined, nonpathogenic cellular system.

15 cl, 45 dwg, 2 tbl, 20 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

Up!