Method for commercial production and purification of human recombinant growth hormone of inclusion bodies

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

3 dwg, 2 tbl, 4 ex

 

The invention relates to the field of biotechnology, in particular to the industrial method of obtaining recombinants of human growth hormone (rgrc) person or somatropin.

The present invention is the development of technology is easily scalable and cost-effective method of producing high-purity rgrc, with a purity not less than 98%.

Growth hormone (somatotropin-STG) is a protein hormone secreted by the anterior pituitary gland. Secreted into the blood under the influence of somatostatin and STG-releasing factor - somatoliberin. Time and frequency allocations are regulated by somatostatin, and the amount of HGH is regulated by somatoliberin. HGH refers to the group of anabolic hormones [Pavlovsky A.G. and others // the human growth Hormone. Collection of scientific papers. Research center of biological research of the Academy of Sciences of the USSR, Pushchino, 1988., So 305, No. 4., S-864.]. The HGH molecule composed of 191 amino acid residue with a molecular mass of 22125 Yes. Keeping the scheme structure common to other mammals (contains two tryptophan residue, three tyrosine residue and four cysteine residue, which form in the molecule two disulfide bridge to form two loops - large, including the Central plot of amino acid sequence between Cys54-Cys165and small (on the C - terminal site) between Cys82 -Cys189), HGH differs in amino acid sequence from the studied somatropin animals at 34-35%. This explains the inefficiency of somatropin animal as a growth promoter when administered to humans [Y.A. Ovchinnikov // Bioorganic chemistry. M: Education, 1987, s.].

Recombinant human growth hormone is widely used in medical practice for the treatment of various diseases associated with decreased secretion of this hormone in the human body - makorokoto syndrome Shereshevsky-Turner, pituitary nanism [Abdelaziz Elamin, Tuvermo A. // Growth hormone treatment in children and adolescents. Annals of Saudi Medicine, 2002, V.22, P.47-55]. According to scientific studies, rgrc can be used in complex therapy of diseases such as type II diabetes, hypertension, atherosclerosis, cardiovascular disease, obesity and other pathologies that occur as a result of lower levels of HGH in the human body with age [Wilson M.E. Ter-Minassian, Gordon TR, Chikazawa, K., Gust D., Tanner J.., Rudman .G. // Effects of growth hormone on neonatal growth in nursing rhesus monkeys. J. Clin. Endocrinol. Metab., 1991, V.72(6), P.1302-1307.].

The creation of producer strain HGH allowed to obtain biologically active protein in the quantity required for various structural and functional studies and for use in medical practice. Created abroad medicinal p is aparaty based rgrc (Genotropin, protropin, humatrop, and others). Growth hormone is produced in bacterial cells of E. coli, contains an additional N-terminal residue of methionine (Met-rgrc), which must be split to obtain a desired drug (rgrc).

Thus obtained rgrc purified by standard methods mentioned, for example, in U.S. patents: US 4601980, US 5424199, US 4658021 and others In these ways usually, the solubilization of the protein in buffer systems containing chaotrope agents or ionic detergents in high enough concentrations, which seriously hinder the protein purification in the subsequent stages. Also known way for solubilization rgrc with urea at high pH values [Ashok K. Patra et all // Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli, Protein Expression and Purification, 2000, V 18, p.182-192]. Resaturate protein is carried out by lowering the concentration chaotropes agent or the addition of a redox pair. Further purification rgrc carried out by the following methods: fractionation by polyethylenimine, gel-filtration on Sephacryl S-200, fractionation with ammonium sulfate, ion exchange chromatography, affinity chromatography using antibodies against growth hormone. Such methods are time consuming and expensive.

There are also known methods of obtaining rgrc, [U.S. Pat. EP NO. 0114506, THE F 1596731, RF 2099348]in which they proposed to carry out the purification of the protein using the precipitation of various salts of metals, ensuring, thus, disposal of the product from the high molecular weight protein contaminants. But this approach leads to long-term processes of centrifugation or filtration. These methods complicate the process of protein purification and increase the cost of the technology.

Closest to the claimed method is a method [Vorob'ev A.I., etc. //Highly efficient process of obtaining human growth hormone, Biotechnology, 2005, No. 5, P.44-48], allowing to obtain intact native rgrc, which consists of several stages: solubilization Taurus include using 6 M guanidine hydrochloride, holding refolding by adding to the reaction mixture of 1 mm group probably facilitates and 0.1 mm applied, the protein purification on ion-exchange sorbent Source 15Q, cleavage of N-terminal methionine with the amino peptidases. Final purification was performed using reverse-phase chromatography sorbent C4, Nucleosil. For protein translation in pharmacological buffer solution used exclusive gel-filtration on a Superdex gel 75. This method allows you to get rgrc with the release of 0.1 g of 1 g of the target protein, solubilizing of Taurus enabled.

The disadvantage of the prototype method is that using 6 M guanidine hydrochloride for when globalizatsii reduces the release of Met-rgrc after renaturation process and leads to large losses of protein up to 50% of the initial amount. Another disadvantage of this method is the need for protein concentration after renaturation by deposition of a 50% solution of ammonium sulfate, which in turn when scaling process leads to large losses of protein and complexity at the stage of centrifugation.

Thus, there is a need for new and improved and cheaper ways of cleaning rgrc, which allows to obtain a highly purified protein with high yields, and reduce cleaning time.

The technical result of the claimed invention is the simplification of the purification of recombinant human growth hormone.

This technical result is achieved by carrying out the claimed method of purification of recombinant human growth hormone. The proposed method of obtaining and purification rgrc synthesized in the cells of E. coli as insoluble Taurus turn, consists of several consecutive stages.

Bullock include Met-rgrc solubilizer in a buffer solution containing 20 mm Tris-HCl, 2 M urea, pH 11.0 and allowed to mix at room temperature.

The protein solution obtained after solubilization, diluted with a buffer solution containing 20 mm Tris-HCl. Then bring the solution pH to a value of 8.0, add 37% N2About2and continue to mix at room temperature is. The number of correctly laid protein monitored by reverse phase HPLC on a column of YMC C4, 250×4,6, 5 μm, 300 A (YMC Co., LTD, Japan).

The first phase chromatographic purification of Met-rgrc performed at room temperature on the axial column Packed with ion-exchange sorbent Q Sepharose FF (GE Healthcare, USA). The protein solution after renaturation applied to the column equilibrated starting buffer solution containing 20 mm Tris-HCL, pH 8.0. Elution of the protein from the column are linear concentration gradient of sodium chloride from 0-40%.

After the first stage of purification on Q Sepharose FF to get the native protein (rgrc) carry out the cleavage of N-terminal methionine. To do this, in solution Met-rgrc add lacinova aminopeptidase. The reaction of cleavage was performed at room temperature and monitored by means OF isocratic HPLC on a column of Symmetry 300, C18, 300 Å of 4.6×250 mm) (Waters, USA).

In the second phase chromatographic purification rgrc, protein derived methionine put on the axial column, Packed Butyl Sepharose 4 FF (GE Healthcare, USA) and equilibrated starting buffer solution containing 20 mm Tris-HCL, 2M NaCl, pH 7.0. Elution of the protein with the column conducting a declining linear concentration gradient of sodium chloride from 100% to 0.

After hydrophobic chromatography spend a third phase chromatographic purification rgrc on the axial columns is, Packed ion exchange sorbent Q Sepharose FF (GE Healthcare, USA). The protein solution was transferred by gel-filtration on Sephadex gel G-25 Fine (GE Healthcare, USA) in a buffer solution containing 50 mm Histidine, pH 6.5. Then the obtained protein solution applied to the column with ion exchange sorbent, balanced starting buffer solution containing 50 mm Histidine, pH 6.5. Elution of the protein from the column are linear concentration gradient of sodium chloride from 0-30%.

The final stage chromatographic purification rgrc spend on a column filled with a gel Sephacryl S-100 HR (GE Healthcare, USA). Pre-column balance buffer solution containing 20 mm Na2HPO4, 0.2% glycine, 4% mannitol, pH 6,8.

The result is 0.45 g rgrc with purity by HPLC 98-99% of 1 g of the target protein obtained from Taurus enabled.

Application of the proposed invention has the following advantages.

The solubilization Met-rgrc carried out in 2 M urea at pH 11.0, which allows you to save the formed secondary structure of a protein during the formation Taurus inclusion in the cell is E. coli. The use of a higher pH leads to the conversion of the disulfide bond Cys182-Cys189in thioester linkage, which in turn entails the formation are difficult to separate in terms of industrial production related impurities and the reduced activity of the target product.

- The process of renaturation of a protein is carried out at a concentration of 1.2 mg/ml, allowing this stage in small amounts. As oxidant sulphhydryl groups of the protein for the first time use hydrogen peroxide. Under these conditions, during the renaturation process was reduced to 2 h at industrial scale production.

Thanks to proper conditions, the yield of the target protein after renaturation was raised to 75-80%.

- Completely excluded stage of concentration of the protein before chromatographic purification via deposition of protein 50% solution of ammonium sulfate.

- Stage protein purification on reversed-phase sorbent replaced by a purification step using hydrophobic chromatography, which resulted in a significant simplification and cheapening of the process.

The invention is illustrated by the following figures.

Figure 1 shows the amino acid sequence rgrc.

Figure 2 presents the control of the removal of N-terminal methionine rgrc with reverse phase HPLC

Figure 3 presents the analysis rgrc method OF HPLC after solubilization, renaturation.

The invention is illustrated by the following examples.

Example 1. Obtaining high-purity rgrc.

To 200 g of wet Taurus enable rgrc poured 4 l of buffer solution containing 20 mm Tris-HCl, 2 M urine is ins, pH 11.0, and left to mix for 30 min at room temperature. The protein solution obtained after solubilization, diluted with a buffer solution containing 20 mm Tris-HCl. Bring the solution pH to a value of 8.0, add 37% H2O2. Then the reaction mixture was allowed to mix at room temperature for 2 hours.

The number of correctly laid protein monitored by reverse phase HPLC on a column of YMC C4. The first phase chromatographic purification rgrc performed at room temperature on the axial column series BPG 140×500 mm, filled with ion exchange sorbent Q Sepharose FF. The protein solution after renaturation applied to the column equilibrated starting buffer solution containing 20 mm Tris-HCL, pH 8.0. The load on the sorbent can achieve 80 mg total protein per 1 ml of media. After application of the total volume of protein a sorbent is washed with three volumes of starting buffer solution. Elution of the protein with the column conducting a linear concentration gradient of sodium chloride from 0 to 40%. Detection of protein was performed at the wavelength equal to 280 nm. The fractions containing the target protein, the results of the analysis on a reversed-phase column (C4join.

In the next step, to a solution of Met-rgrc obtained after purification on Q Sepharose FF add lacinova aminopeptidase in at 7.5% on 1 Galka. The reaction removal carried out for 16 h at room temperature. Reaction control using isocratic RP HPLC on a column of Symmetry 300, C18, a 4.6×250 mm, 5 μm, 300 A (Waters, USA), 2.

After removal of N-terminal methionine chromatographic purification rgrc was performed using hydrophobic chromatography at room temperature on the axial column series BPG 140×500 mm, filled with Butyl Sepharose 4 FF. To the protein solution after ion exchange chromatography add sodium chloride to a final content of 2 M, bring the pH to a value of 7.0 and applied to the column equilibrated starting buffer solution. After application of the total volume of protein a sorbent is washed with three volumes of starting buffer solution, which contains 20 mm Tris-HCL, 2M NaCl, pH 7.0. Elution of the protein with the column conducting a declining linear concentration gradient of sodium chloride 100 to 0%. Detection of protein was performed at the wavelength equal to 280 nm. The fractions containing the target protein, the results of the analysis on a reversed-phase column With a4join.

The combined fraction of translated protein by gel-filtration on Sephadex gel G-25 Fine in a buffer solution containing 50 mm Histidine, pH 6.5. Then the obtained protein solution applied to the column with ion exchange sorbent Q-Sepharose FF equilibrated starting buffer solution containing 50 mm Gisti the ina, pH 6.5. Elution of the protein from the column are linear concentration gradient of sodium chloride from 0-30%. Detection of protein was performed at the wavelength equal to 280 N. M. the Fractions containing the target protein according to the results of the analysis on a reversed-phase column With a4join.

The final stage chromatographic purification rgrc spend on a column filled with a gel Sephacryl S-100 HR. Pre-column balance buffer solution containing 20 mm PA2NRA4, 0.2% glycine, 4% mannitol, pH 6.8. Then hold gel-filtration of the obtained protein solution after the third stage of the chromatographic purification. Detection of protein was performed at the wavelength equal to 280 nm. In the obtained fractions of the protein concentration is determined using reversed-phase chromatography on a column of YMC, C4.

The product yield was 0.45 g of high-purity rgrc with 1 g of the target protein, obtained by solubilization of the Taurus inclusion. The purity of protein - not less than 98%. The material balance of an industrial cleaning process rgrc presented in table 1.

≤1
Table 1
The material balance of the main stages of the industrial process cleaning rgrc
The STAGE of PURIFICATION of RG is H OUTPUT rgrc, gOUTPUT rgrc, %Chromatographic purity rgrc,%CONTENT
Proteins of E. coli, ng/mgproteinEndotoxins, U/mgprotein
Extraction of protein
20 grams of Taurus enable40100->250>250
Resaturate30-3275-8075-80
Ion-exchange
chromatography on26-3166-7785-90 100-250
Q Sepharose FF, rn,0
Hydrophobic chromatography on
Butyl Sepharose 423-2558-63
89-95
FF Gel filtration
on Sephadex G-2522-2355-59
Ion-exchange1-3
chromatography on
18-2046-5095-99
Q Sepharose FF, RS,5
Gel filtration
on Sephacryl S-100HR17-1844-4797-99

Example 2. Control removal of N-terminal methionine rgrc by reverse phase HPLC on a column of Symmetry 300, C18.

Chromatography is carried out in isocratic mode on a reversed-phase column (Symmetry 300, C18, 5 μm, 300A, and 4.6 × 250 mm) (Waters, USA). The mobile phase contains 0.5 M KN2RHO4pH 6.5, 24% of 1-propanol. The flow rate is 0.5 ml/min, temperature - 45°C, wavelength 220 nm. the external standard using the European standard (CRS) of known concentration. The product eluted as one peak (figure 2), the purity of the product is at least 98%.

Example 3. Analysis rgrc by reverse phase HPLC on a column of YMC C4.

Analysis of the obtained rgrc performed using reversed-phase chromatography in a gradient concentration of 1-propanol:acetonitrile (20:80) from 30 to 50% in the presence of 0.1% TFU on a column of YMC C4, H,6 mm, 5 μm, 300A (YMC Co., LTD, Japan) on the chromatograph Agilent 1200 series (Agilent Corporation, USA). The product eluted as one peak (figure 3), the purity of the product is at least 98%.

Example 4. The scale purification process somatropin rgrc.

The data are shown in table 2 were obtained with the 10 pilot batches of the drug.

Table 2
The CLEANUP PHASE rgrcOUTPUT rgrc, gOUTPUT rgrc, %Chromatographic. purity rgrc, %CONTENT
Proteins of E. coli, ng/mgproteinEndotoxins, U/mgprotein
Extraction of protein
from 2000 Taurus enable400100->250>250
Resaturate300-32075-8075-80
Ion-exchange
chromatography on Q Sepharose FF265-31066-7785-9050-100100-250
Hydrophobic chromatography on
Butyl Sepharose 4235-25058-63
FF89-95
Gel filtration
on Sephadex G-25220-23755-59
Ion-exchange1-3≤1
chromatography on Q Sepharose FF187-20046-5095-99
Gel filtration
on Sephacryl S-100 HR172-18544-4797-99

1. The method of obtaining and purification of recombinant human growth hormone, comprising the following stages:
1) the dissolution of bodies is C included in 2M urea at pH 11;
2) resaturate protein in a buffer solution of 20 mm Tris-Hcl at pH 8.0 using as oxidant sulphhydryl groups of protein hydrogen peroxide;
3) ion exchange chromatography on Q Sepharose FF, pH 8.0;
4) cleavage of the N-terminal methionine using latinboy amino peptidases;
5) hydrophobic chromatography on Butyl Sepharose 4 FF;
6) gel filtration on Sephadex G-25;
7) ion-exchange chromatography on Q Sepharose FF, pH 6.5;
8) gel filtration on Sephacryl S-100HR.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: disclosed is a binding protein which is specific to IL-13, having 6CDR sections (three light and three heavy chains). The invention discloses a structure of an antibody based on this protein and a conjugate based on the antibody. The invention describes versions of nucleic acid which codes the antibody or structure, and an expression vector, a replication vector and cells bearing such vectors. Described is a method of producing the protein by culturing cells and proteins obtained using said method. Described are compositions based on versions of the proteins.

EFFECT: invention can be used in medicine to treat and diagnose diseases associated with IL-13 activity which negatively affects health.

50 cl, 23 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: producing protein is ensured by bacterial host cell culture containing a recombinant nucleic acid in a medium containing crude glycerol as a carbon source, and protein recovery expressed by the cell. Crude glycerol represents a side product of bio fuel or soap which is found in the medium in the concentration of 0.1% to 75% (vol./vol.). The culture process is performed in an enclosed volume, injection or continuous fermentation. The bacterial cell is specified in Streptomyces Hvidans, Bacillus subtilis and Streptomyces rubiginosis.

EFFECT: higher accumulation of cell biomass and protein production in the culture.

11 cl, 14 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: for the purpose of producing a probiotic preparation, a biomass of the symbiontic strain Corynebacterium diphtheriae tox No. 2 of Federal State Institution L.A. Tarasevich State Institution of Standardization and Control is cultured in a liquid nutrient medium; microbial cells are deposited by centrifugation, washed to precipitate whole cells by centrifugation; the cells are suspended in a sodium chloride solution. Then, the cells are disintegrated at temperature 25-30°C for 15 min at frequency 20 kHz and amplitude 14 mcm; the desintegrant is centrifugated at 5000 g; and the remained whole cells are removed. The prepared precipitation is centrifugated at 14000 g for 20 min. to produce precipitated corpuscular antigens of cell walls of lipopeptidopolysaccharide corynebacteria which is resuspended and disintegrated at temperature 25-30°C for 5 min. at frequency 20 kHz and amplitude 14 mcm. Then the preparation is diluted to the concentration of 225-275 mcg/ml, sterilised and lyophilised.

EFFECT: use of the invention enables producing the safe and effective preparation of corpuscular antigens providing creating specific and non-specific immunity to tuberculosis.

2 cl, 2 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to obtaining and application of staphylococcal anatoxin-vaccine for prevention and treatment of animal diseases of staphylococcal etiology. Method by invention includes growing staphylococci on synthetic nutritional medium with further autoclaving of obtained suspension of microorganisms. After that, detoxication of obtained complex of staphylococcal exo-, endo- and superenterotoxins is performed by two detoxicators: first by 0.2-0.3% glutardehyde solution during 3-5 days at 40-42°C, then by 0.2% ethonium solution or 0.15-0.25% solution of alkylmethylbenzyl ammonium during 3-5 days at 40-42°C. After that, target product is sorbed on aluminium hydroxide in dose 3-5 mg/ml.

EFFECT: method application makes it possible to increase protective and therapeutic efficiency and safety of anatoxin-vaccine.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology. The invention discloses a composition for coexpression in an eubacterial host cell orthogonal tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synthetase (O-RS), which preferably aminoacylates said O-tRNA with an unnatural amino acid. The disclosed composition consists of two nucleic acid constructs: the first construct contains promoter and terminator nucleotide sequences derived from an E.coli proline tRNA gene and which is derived from the archaea of the expressed sequence, which encodes one O-tRNA or is a polycistronic operon, and the second construct contains a modified E.coli glnS promoter and the expressed nucleotide sequence which encodes the corresponding O-RS. Described is a translation system which includes the disclosed vector constructs, and a method of obtaining the polypeptide of interest which contains an unnatural amino acid in a genetically defined position.

EFFECT: obtaining properties with new properties, which are defined by inclusion of unnatural amino acids into a predetermined position.

54 cl, 8 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for providing syalidase enzymatic activity in a cell line culture on the basis of cell transfection by a polypeptide expression vector having syalidase activity and containing a sequence coding a catalytic domain of the enzyme syalidase with a lower number of sialic acid residues wherein polypeptide is secreted into a medium together with Fc-containing protein. What is also presented is a related vector. The invention is applicable in treating diseases and conditions requiring the control of affinity to one or more FcγRI, FcγRllA and FcγRIIIA receptors, ADCC activity, macrophague or monocyte activation, half-life in a serum and avidity.

EFFECT: higher effectiveness of the use of the invention.

14 cl, 14 dwg, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining factor VII (FVII) and factor Vila (FVIIa) albumin linked polypeptides, and can be used in medicine. A polypeptide, which is a FVII or FVIIa polypeptide is obtained in a recombinant manner, said peptide being linked with albumins through a glycerine-serine peptide linker of a special structure, which separates part associated with FVII or FVIIa from the albumin part, wherein the FVII or FVIIa polypeptide lies on the N-end of the fused protein. The linked polypeptide or vector structure, which contains its coding nucleic acid, is used as a medicinal agent for treating or preventing blood-clotting disorders.

EFFECT: invention enables to obtain a protein with FVII or FVIIa biological activity and longer functional half-time in plasma in vitro compared to non-linked FVII or FVIIa.

12 cl, 4 dwg, 6 tbl, 6 ex

FIELD: medicine.

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

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

2 cl, 5 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: hybrid polypeptide is a polypeptide 1 to which a polypeptide 2 is covalently bonded, where polypeptide 1 is a human endostatin sequence with 135-184 amino acid residues, wherein at position 173, Cis is replaced with Ala with a relatively native endostatin sequence, and polypeptide 2 is a human plasminogen sequence with 82-341 or 463-511 amino acid residues. Also disclosed is a method of obtaining the hybrid polypeptide using an E.coli producer, which involves methods for extraction and purification thereof.

EFFECT: polypeptide is capable of inhibiting human endothelial cell proliferation in vitro and can be used when producing nontoxic preparations for inhibiting angiogenesis.

4 cl, 4 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: what is disclosed is a nucleic acid molecule which codes F-protein of respiratory syncytial virus or a fragment of said protein optimised for expression in a human cell. There are also disclosed a vector and a composition which contain said nucleic acid molecule.

EFFECT: invention may be used for producing a respiratory syncytial virus vaccine to be used in medicine.

8 cl, 15 dwg, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a polypeptide and its homodimer which possess agonist activity with respect to a growth hormone receptor, a nucleic acid molecule coding them, a vector which involves said nucleic acid, a cell of expression of said polypeptide, a pharmaceutical composition and a method with the use of said polypeptides for treating growth hormone deficiency. The polypeptide has an amino acid sequence presented in SEQ ID NO: 11 or 12. The homodimer consists of two polypeptides consisting of SEQ ID NO: 11 or 12. The nucleic acid molecule consists of a nucleic acid sequence presented in SEQ ID NO: 4. The pharmaceutical composition to be used in treating growth hormone deficiency contains said polypeptide or homodimer and an excipient or a carrier. The method of treating growth hormone deficiency involves introducing an effective amount of said polypeptide or homodimer.

EFFECT: invention provides creating the polypeptide which possess agonist activity on growth hormone receptor, and it is effective in treating growth hormone deficiency.

17 cl, 9 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining a modified growth hormone, and can be used in medicine. By recombination, a polypeptide is obtained, which has antagonistic effect on the growth hormone receptor.

EFFECT: invention enables to obtain a polypeptide which is effective when treating conditions caused by excess growth hormone in the body of the patient.

11 cl, 19 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: there is offered a method of engineering yeast strains - stable human growth hormone (somatotropin) producers. Also, there are offered two strains Y-3506 of Russian National Collection of Industrial Microorganisms and Y-3507 of Russian National Collection of Industrial Microorganisms - stable human somatotropin producers. The producer strains have been prepared by sequential integration of expression plasmid into a recipient stain genome. Each plasmid carries in its structure a somatotropin gene (GH1); fused with a leader sequence controlled by the GAL1 promotor, as well as one of the genes URA3, LEU2, TRP1 and HIS3 complementing auxotrophic recipient strain mutations.

EFFECT: efficacy of the produced strains is 100-130 mg of somatotropin per 1 l of the medium.

3 cl, 7 dwg, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: proposed are human growth hormone conjugates, obtained by removing a hydrogen atom from -NH2 in the Gln side chain which is formed from the human growth hormone or a human growth hormone compound.

EFFECT: design of an efficient method of producing human growth hormone conjugates.

6 cl, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.

EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.

35 cl, 16 dwg, 1 tbl

The invention relates to biotechnology and can be used to obtain a properly curled, containing the precursor of insulin chimeric protein

The invention relates to a peptide that includes the analogue of carboxykinase sequence of growth hormone, where carboxykinase sequence contains amino acid residues 177-191 human growth hormone: Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe, or a corresponding sequence of growth hormone, non-human mammal; where in the specified similar amino acids at positions 182 and 189 hGH are connected by a communication in order to facilitate the formation of cyclic conformation, and/or amino acids at positions 183 and 186 hGH are connected by a salt bridge or a covalent bond; or its salts with organic or inorganic acid

The invention relates to an improved method for producing adducted end of the condensation products, which are Schiff bases, components of which include a protein having useful activity in animals, and aromatic o-hydroxyaldehyde, which connect the above components in an aqueous medium at pH 7.0 or higher to form a reaction mixture under conditions effective to conduct specified the condensation reaction essentially to completion by using the stage of fast compared to drying in ambient conditions removal 97,0 to 99.9% by weight, preferably approximately 98,0 - 99,0% by weight of water, already present or formed during this reaction, condensation, consistent with maintaining the integrity of reagents condensation and adductor final product

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to an isolated decapeptide or nonapeptide, capable for inducing cytotoxic T cells, as well as said peptides in which 1 amino acid is substituted, a polynucleotide which codes said peptides, a pharmaceutical composition and a vaccine, which contain said peptides, a method of inducing antigen-presenting cells, a method of inducing cytotoxic T cells, an isolated cytoxic CD8+ T cell, a dendritic cell which induces CTL and a method of treating a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes. The isolated decapeptide or nonapeptide, which is capable of inducing cytotoxic T cells, contains amino acid sequences selected from a group consisting of SEQ ID NO: 7, 8, 12, 9, 10, 11, 192, 195, 197, 209, 225, 226, 228, 230, 240, 241, 243, 244, 253, 254 and 255. The pharmaceutical composition for treating a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes contains one or more said peptides and a pharmaceutically acceptable carrier. The method of treating a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes in an individual involves administering to the said individual a vaccine containing one or more said peptides.

EFFECT: invention enables to obtain peptides which are used to treat a disease associated with high expression of SEQ ID NO: 1, 3 and/or 5 genes.

22 cl, 26 dwg, 7 tbl, 2 ex

Up!