Producing il-21 in prokaryotic host cells
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
The text descriptions are given in facsimile form.
1. Expression vector for production in E. coli protein IL-21, containing the following functionally connected elements:
(a) a replication origin;
(b) a DNA element of transcription initiation;
(c) the polynucleotide sequence presented as SEQ ID NO:27, and
(d) a transcription terminator.
2. The expression vector according to claim 1, which further comprises breeding the token.
3. Expression vector rtar for production in E. coli the protein encoded by the nucleotide sequence presented as SEQ ID NO:27, which are deposited in ATSC number of MOUTH-4853.
4. A host cell E. coli transformed with the expression vector according to any one of claims 1 to 3.
5. A host cell according to claim 4, which is a cell strain E. coli W3110.
6. The method of producing the protein of IL-21, including:
(a) to livelounge host cells according to claim 5 in environment, suitable for the expression of IL-21;
(b) removing cells from the environment for cultivation;
(c) isolation of cell protein IL-21.
7. The method of producing the protein of IL-21, including:
(a) culturing host cells according to claim 5 in environment for growing periodic fermentation injection;
(b) removing cells from the environment for cultivation;
(c) isolation of cell protein IL-21.
8. The method of producing the protein of IL-21, including:
(a) culturing host cells according to claim 4 or 5 in shake flask until OD600 of 5 to 20 in the medium for cultivation;
(b) seeding apparatus for fermentation from 1 to 12% vol./about. the contents of the shake flask;
(c) culturing the cells in the apparatus in the growing medium at pH from 6.2 to 7.2, in which nutrient solution previously served 15 h actual duration of fermentation (EFT);
(d) adding 20-30 hours actual duration of fermentation inducing agent;
(e) collection of host cells in the range from 48 to 56 hours of actual duration of fermentation;
(f) isolation of cell protein IL-21.
9. The method according to claim 8, in which the inducing agent is isopropylcyclopentadienyl (IPTG) at the rate of 0.5 to 2 mm.
10. The method of claim 8, where the nutrient solution contains a carbohydrate selected from the group consisting of glycerol and glucose at a concentration of media for growing and velocity Pete the Oia 5-15 grams of carbohydrate per hour.
11. The method according to claim 10, where the glycerol is 40-70% (vol./about.) glycerol and glucose is 40-70% (wt./about.) glucose.
12. The method according to claim 10, where the content of glycerin is about 70% (vol./about.) or the glucose content is about 60% (wt./vol.).
13. The method of producing the protein of IL-21, including:
(a) preparation of seed flasks with inoculum containing cell host according to claim 5, and with culture medium containing approximately 5 g/l glycerol;
(b) culturing the inoculum in the environment for growing for 16-20 h at approximately 30°C;
(c) the transfer of the cultured supernatant in the culture medium in the fermentor for periodic cultivation in a concentration of 0.5-5% (vol./vol.);
(d) fermentation periodic cultivation at about 37°C. and pH of about 6.8 in the presence of approximately 2% glycerol;
(e) commencement of injection at 8 o'clock the actual duration of the fermentation of glucose supply with parameters corresponding to about 9.5 g/l/h, and continuing this procedure until the expiration time of fermentation;
(f) adding at the 24 hour IPTG to a final concentration of from 0.5 to 2 mm;
(g) the fermentation is approximately 28 h after addition of IPTG;
(h) discharge of fermentation medium from the fermenter;
(i) adding to the fermentation medium is approximately equal volume of water;
(j) homogenization and centrifugation, the enzyme is operating environment for the collection of sediment or suspension cells, containing protein material;
(k) the selection of cell protein IL-21.
14. A host cell strain, designated ZGOLD1 and deposited in ATSC number of MOUTH - 5698, which transformed vector rtar described in claim 3, producing IL-21.
13.12.2002 according to claims 1-14.
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
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: medicine; biotechnologies.
SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.
EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.
FIELD: medicine, biotechnologies.
SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.
EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.
20 cl, 2 dwg, 8 ex
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to biotechnology. Method includes addition to fermentation broth or homogenate from E. coli of efficient quantity of ethacridinlactate solution for sedimentation of contamination from host-cells in conditions, when greater part of polypeptide remains dissolved, and isolation of heterological polypeptide from broth or homogenate.
EFFECT: simplification of target polypeptide purification and obtaining it with high degree purity.
23 cl, 15 dwg, 3 tbl
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.
EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.
2 cl, 7 dwg
SUBSTANCE: invention concerns biotechnology, specifically production of new polypeptides regulating carbohydrate metabolism, and can be used in medicine. New polypeptides reacting as both GLP-1 receptor agonists and glucagon receptor antagonists. Polypeptides and coding nucleic acids are used as components of pharmaceutical compositions for treatment of diabetes type 2 and metabolism disorders.
EFFECT: production of compounds providing effective glucose homeostasis for patients, suffering from carbohydrate metabolism disorders.
18 cl, 1 dwg, 4 tbl, 20 ex
SUBSTANCE: invention relates to genetic engineering, namely, to obtaining inhibitors of TGF-β1 and can be used in medicine. Obtained peptides are capable of binding with transforming growth factor TGF-β1 and are potential inhibitors of biological activity of TGF-β1, binding with this cytokine directly. Peptides are obtained by recombinant method using transformed host-cell, by cultivating host-cell in conditions which ensure production of the said peptide, and its separation. The invention allows for efficiently treatment of diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.
EFFECT: possibility to efficiently treat diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.
12 cl, 6 dwg, 4 tbl, 4 ex
FIELD: biotechnology, in particular provision storage.
SUBSTANCE: bacteriocin represents polypeptide isolated from lactobacillus sakei 2512 and is capable to suppress lysteria growth and reproducing. Bactericin has specific amino acid sequence represented in claims. Nuclear acid sequence encoding said polypeptide is disclosed. Also disclosed is a vector including nuclear acid sequence for cloning and/or expression of polypeptide, for example in transformed cells, selected from Lactococcus, Lactobacillus, etc. Method for production of recombinant polypeptide is developed. Claimed bactericin or strain 2512 are used as component of bactericide composition, being capable to suppress growth of gram positive pathogenic bacteria, in particular Listeria monocytogenes.
EFFECT: large scale application of bacteriocin against pathogenic or undesired flora in food industry.
13 cl, 2 dwg, 1 tbl, 3 ex
FIELD: chemistry, genetics.
SUBSTANCE: invention relates to the field of genetic engineering and can be used for obtaining human interleukin-13 (IL-13). On the basis of plasmid pTrcTEGF and optimised for bacterial expression cDNA encoding a mature form of human interleukin-13 a recombinant plasmid DNA pTrcTREN-IL13 is constructed. This plasmid DNA provides biosynthesis of polypeptide with properties of human interleukin-13 in cells of E.coli. As a result of transformation of strain of bacteria Escherichia coli BL21(DE3) by the said plasmid, strain of E.coli BL21(DE3)/pTrcTREN-IL13/pTrcTREN-IL13 is obtained, which produces polypeptide with properties of human interleukin-13.
EFFECT: ensuring an increase in the level of heterological expression of polypeptide with properties of human interleukin-13 at reduced quantity of inductor.
2 cl, 4 dwg, 1 tbl, 4 ex
FIELD: molecular biology, medicinal genetics.
SUBSTANCE: the present innovation could be applied in clinical practice for predicting the flow of chronic hepatitis C (CHC). It has been suggested a predictive model that enables to evaluate the risk for the development of hepatic fibrosis (development of cirrhosis) in case of CHC. The present model is based upon the sum of the results of DNA assay in patients for the presence of polymorphisms of cytokins genes(-511)C/T IL-lb, (-174) G/C IL-6 gene and (+915) G/C TGF-bl gene. Moreover, it has been established that genotypes (-511) TT IL-lb gene, (-174) CC IL-6 gene and (+915) GC TGF-bl gene are characterized with pronounced "profibrotic" action; genotypes (-511) CT IL-lb gene and (-174) GC IL-6 gene - with moderate "profibrotic" action; genotypes (-511) CC IL-lb gene, (-174) GG IL-6 gene - with protective action concerning the development of cirrhosis, and genotype (+915) GC TGF-bl gene is being neutral. The present innovation enables to detect the high-risk group of cirrhosis development in CHC-patients that needs intensive antiviral and in future - in antifibrotic therapy.
EFFECT: higher accuracy of prediction.
5 dwg, 6 ex, 11 tbl
FIELD: biotechnology, in particular plant gene engineering.
SUBSTANCE: recombinant plasmid DNA pBi101-IL18 is constructed having length of 15000 n.p. and containing DNA fragment of pBi101-IL18 vector plasmide having length of 13900 n.p.; -NdeI-BamHI/BgIII DNA fragment being cDNA site of human IL-18 gene; -Nol-NdeI fragment of pET15b plasmide; encoding N-terminal polypeptide from six hystidine amino acids and site of thrombin enzyme hydrolysis, -5'URT region from genome of tobacco etch virus; double 35S CaMV promoter from genome of cauliflower mosaic virus; -3'URT region from genome of cauliflower mosaic virus. Method of present invention makes it possible to transfer nucleotide sequence of human IL-18 in plant genomic DNA.
EFFECT: method for production of mature human IL-18 having biological activity.
2 dwg, 1 tbl, 3 ex