METHOD OF RECOMBINANT HUMAN INSULIN PREPARING

FIELD: biotechnology, preparative biochemistry, endocrinology. SUBSTANCE: the strain-producer E. coli JM109/pPINS07 is cultured, cells are disrupted by disintegration, inclusion bodies containing the fused protein are dissolved in a buffer containing urea and dithiothreitol, renaturated and the fuse...

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Hauptverfasser:	URAKOV N.N, DERBYSHEV V.I, DONETSKIJ I.A, GORKUN O.G, LIPIN M.JU, KOROBKO V.G, VOROTNIKOVA I.I, NOZDRIN V.N, KRASIL'NIKOV V.A, BEKMURATOVA I.I, STEPANOV A.V, KOBELEVA V.A, DUNAJTSEV I.A, GAVRIKOV V.G, KOSTROMINA T.I, MARTOVETSKIJ M.N, FEDJUKIN V.S, SHEPELIN A.P, MIROSHNIKOV A.I, KALININ JU.T, IVANOV V.T, KOROBOV V.I, BORISOV N.V, ABROSIMOVA L.I, BAJDUS' A.N, GULJAEV V.A, LITVINENKO M.A, BAIRAMASHVILI D.I, SHMATCHENKO N.A
Format:	Patent
Sprache:	eng
Schlagworte:	BEER BIOCHEMISTRY CHEMISTRY COMPOSITIONS THEREOF CULTURE MEDIA ENZYMOLOGY FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE METALLURGY MICROBIOLOGY MICROORGANISMS OR ENZYMES MUTATION OR GENETIC ENGINEERING PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS SPIRITS VINEGAR WINE
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Beschreibung
Zusammenfassung:	FIELD: biotechnology, preparative biochemistry, endocrinology. SUBSTANCE: the strain-producer E. coli JM109/pPINS07 is cultured, cells are disrupted by disintegration, inclusion bodies containing the fused protein are dissolved in a buffer containing urea and dithiothreitol, renaturated and the fused protein is purified by precipitation of inert compounds in 40% isopropyl alcohol followed by chromatography on CM-Sepharose. Then the fused protein is cleaved by trypsin, products of trypsinolysis are subjected for chromatography on SP-Sepharose using the gradient linear elution with sodium chloride at concentration from 0 to 0.5 M prepared on the initial buffer followed by hydrolysis with carboxypeptidase B. Obtained insulin fraction is purified by method of high-effective liquid chromatography with reversed phase and the following gel-filtration. Method ensures to obtain insulin of the improved quality at purity 96%, not less, and activity 26 U/mg, not less. Method can be used in the industrial scale. EFFECT: improved method of preparing, high purity and activity, availability and safety of sorbents. 1 tbl, 1 ex