METHOD FOR PREPARING RECOMBINANT HUMAN ALPHA-2B-INTERFERON, RECOMBINANT PLASMID AND STRAIN-PRODUCER FOR ITS REALIZATION Russian patent published in 2004 - IPC

FIELD: genetic engineering, biotechnology, medicine, pharmacology.

SUBSTANCE: invention describes new recombinant multicopy DNA plasmid pSX50 encoding synthesis of leukocyte human alpha-2b-interferon being its expression is under control of lactose and tryptophan promoters and transcription terminator. As result of transformation of cells of the recipient strain E. coli BL21 with recombinant DNA plasmid pSX50 the strain E. coli SX50 as a producer of recombinant leukocyte human alpha-2b-interferon is obtained with productivity up to 0.9-1.0 f of alpha-2b-interferon from 1 l of cultural medium. Method for preparing recombinant alpha-2b-interferon is based on using the created recombinant strain E. coli SX50 and involving its submerged culturing in nutrient medium with reduced content of tryprophan at continuous adding nutrient substrates during biosynthesis followed by mechanical disruption of bacterial cells under high pressure, dissolving aggregated protein in the concentrated solution of guanidine hydrochloride and the following renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents and its purification using three-stage chromatography treatment of interferon on resins of type Chelating Sepharose Fast Flow immobilized with ions Cu²⁺, ion-exchange chromatography on ion-exchange resins of type CM Sepharose Fast Flow and gel-filtration chromatography on resins of type Superdex-75. Method provides preparing the interferon substance with purity above 99% by electrophoresis data under reducing and non-reducing conditions, in staining gels with silver, and above 98% by data RF HPLC, and no containing pyrogens (LAL-test) in the amount 400-800 mg from 1 l of cultural medium, not less.

EFFECT: improved preparing method, high yield and purity.

6 cl, 6 dwg, 3 ex