METHOD FOR INDUSTRIAL PREPARING HUMAN RECOMBINANT INSULIN Russian patent published in 2004 - IPC

FIELD: biotechnology, hormones.

SUBSTANCE: invention can be used for preparing human recombinant insulin used for preparing medicinal preparations in treatment of diabetes mellitus. Culturing the strain-producer E. coli JM109/pPINS07 is carried out in industrial fermenter of volume 200-1500 l. The concentration of dissolved oxygen is maintained at the level 40 ± 15%. Cells are disrupted by disintegration and inclusion bodies are dissolved in 8 M urea-containing buffer followed by addition of dithiothreitol. Renaturation of the insulin fusion protein is carried out for a single stage by incubation in 5-10-fold excess of buffer before the purification stage by acid precipitation. Fusion protein is subjected for chromatography on KM-Sepharose. Enzymatic cleavage is carried out successively in the ratio trypsin : fusion protein and carboxypeptidase B : fusion protein = 1 : (500-1000). Between stages of fusion protein cleavage with trypsin and carboxypeptidase B chromatography on SP-Sepharose is carried out. Insulin is purified by method of reversed-phase high-performance liquid chromatography and the following gel-filtration and isolation of the end product in the presence of zinc salts. Invention provides simplifying the preparing highly purified human recombinant insulin and to enhance the yield in the industrial scale.

EFFECT: improved preparing method.

5 cl, 4 tbl, 1 ex