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2 141 531

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Application

99110299/13, 1999-05-26

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Start date

1999-05-26

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Actual filing date

1999-05-26

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Published

1999-11-20

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Inventor

Kalinin Ju.T.Urakov N.N.Ivanov V.T.Stepanov A.V.Miroshnikov A.I.Gavrikov V.G.Bairamashvili D.I.Donetskij I.A.Korobko V.G.Kostromina T.I.Guljaev V.A.Litvinenko M.A.Bajdus' A.N.Korobov V.I.Krasil'Nikov V.A.Borisov N.V.Shmatchenko N.A.Vorotnikova I.I.Gorkun O.G.Derbyshev V.I.Nozdrin V.N.Dunajtsev I.A.Lipin M.Ju.Bekmuratova I.I.Kobeleva V.A.Shepelin A.P.Martovetskij M.N.Fedjukin V.S.Abrosimova L.I.

(73)

Holder

Rossijskoe Aktsionernoe Obshchestvo Otkrytogo Tipa

METHOD OF RECOMBINANT HUMAN INSULIN PREPARING Russian patent published in 1999 - IPC

Abstract RU 2141531 C1

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

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RU 2 141 531 C1

Authors

Kalinin Ju.T.

Urakov N.N.

Ivanov V.T.

Stepanov A.V.

Miroshnikov A.I.

Gavrikov V.G.

Bairamashvili D.I.

Donetskij I.A.

Korobko V.G.

Kostromina T.I.

Guljaev V.A.

Litvinenko M.A.

Bajdus' A.N.

Korobov V.I.

Krasil'Nikov V.A.

Borisov N.V.

Shmatchenko N.A.

Vorotnikova I.I.

Gorkun O.G.

Derbyshev V.I.

Nozdrin V.N.

Dunajtsev I.A.

Lipin M.Ju.

Bekmuratova I.I.

Kobeleva V.A.

Shepelin A.P.

Martovetskij M.N.

Fedjukin V.S.

Abrosimova L.I.

Dates

1999-11-20—Published

1999-05-26—Filed