Purification and activation of recombinant human Prethrombin 2 produced in E. coli = E. coli에 의해 생산된 재조합 사람 프리트롬빈 2의 정제 및 활성화

Abstract

Human prethrombin 2 was purified from a recombinant E. coli cells in which human prethrombin 2 was produced as insoluble aggregates, amounting to 5 to 10% of the total bacterial proteins. Isolation of inclusion bodies and the subsequent solubilization of the insoluble proteins in a denaturing solution containing sodium dodecyl sulfate and dithio-threitol yielded good recovery of prethrombin 2 with at least 75% purity. Prethrombin 2 was further purified by two passages of Sephacryl S-200 gel chromatography. The purified prethrombin 2 was reacted with the polyclonal antibodies against human prothrombin. The N-terminal sequence of the prethrombin 2 was same as that of the native human prethrombin 2. The purified prethrombin 2 was renatured by stepwise removal of the denaturants by slow dialysis. The renatured prethrombin 2 was activated to thrombin by ecarin, a prothrombin activator from Echis carinatus and the thrombin activity generated was detected by a chromogenic assay. This study showed that recombinant prethrombin 2 produced in E. coli could be converted to thrombin.