中国生物工程杂志

Abstract Alfimeprase(ALF) is a recombinantly modified variant of non-hemorrhagic zinc metalloproteinase fibrolase. By digestion with the clone vector p43-alf, the target gene alf was obtained and cloned into the Pichia pastoris expression vector pPICZα A, through high efficiency transformation and Zeocin selection, the recombinant strains of pPICZ?A-alf / GS115 were obtained. After culture parameters of pH value, methanol daily addition concentration, cell density and methanol induction time points were optimized, the production of recombinant Alfimeprase (rALF) reached up to 425 mg/L. By His•Bind chromatography purification, the purity of secreted rALF was as high as 95 %. SDS-PAGE and western blot analysis showed that rALF had a molecular weight of 24 kDa and bound specifically to mouse anti-His• tag monoclonal antibody, activity identification results of the modified fibrin plate method also demonstrated that the secreted rALF had high fibrinolytic activity. Thus the report set up an important foundation for further studies and industrial production of ALF.