蛇毒纤溶酶Alfimeprase在毕赤酵母中的优化表达及活性鉴定

摘要 Alfimeprase(ALF)是蛇毒纤溶酶Fibrolase的N端突变体，有纤溶活性而无出血活性。通过双酶切从克隆载体上获取目的基因alf，并将其克隆至毕赤酵母分泌型表达载体pPICZα A，经高效电转化、Zeocin筛选鉴定及培养条件pH、每天甲醇流加终浓度、菌体生长密度和甲醇诱导时间的优化，获得了重组ALF(rALF)高表达菌株pPICZ? A-alf / GS115，且在优化条件下rALF的最高产量达425 mg/L。通过His?bind柱纯化后，rALF纯度高达95 %，SDS-PAGE和Western blot分析表明rALF的分子量约为24 kDa，且能与一抗特异结合。纤维蛋白平板法纤溶活性鉴定结果进一步说明rALF获得了高活性分泌表达。这即为ALF的深入研究和工业化生产应用奠定了重要基础。

Optimized Expression of snake fibrinolytic enzyme Alfimeprase in Pichia pastoris and its Activity Identification

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.