METHOD OF PRODUCTION OF RECOMBINANT SERINE PROTEASE INHIBITOR OF KING CRAB Russian patent published in 2015 - IPC C12N9/50 C12N9/64 

FIELD: biotechnology.

SUBSTANCE: RNA is isolated from hepatopancreas of king crab Paralithodes camtchaticus and cDNA synthesis of PC inhibitor is performed from total RNA by the method of reverse transcription, which is amplified by polymerase chain reaction with primers, the resulting PCR product containing the PC inhibitor gene, encoding PC inhibitor with amino acid sequence shown on fig. 2, is cloned into an expression vector, followed by transformation into cells of E. coli, the recombinant E. coli strains thus obtained are cultured for no more than 20 hours at 37°C, and the resulting biomass is subjected to centrifugation, washed from residual medium, resuspended in lysis buffer and lysed, the inclusion bodies are isolated from the resulting biomass by sonication, the lysate is resuspended in washing buffer and the inclusion bodies are precipitated again, the resulting inclusion bodies are dissolved in 8 M urea and preliminary purified on sorbent with gradient elution with the buffer containing 8 M urea and 0.7 M NaCl, then renaturation of the dissolved inhibitor is carried out, by the method of reduction of the denaturant concentration in the buffer of 20 mM MES-NaOH pH 6.0 or 20 mM MES-NaOH pH 6.0, 2 M NaCl, 0.05% PEG 6000 and subsequent purification of the preparation by ion exchange chromatography.

EFFECT: invention enables to obtain a serine protease inhibitor having improved inhibitory properties with respect to collagenase, trypsin and subtilisin.

6 cl, 8 dwg, 1 tbl, 2 ex