蛇毒纤溶酶Alfimeprase在毕赤酵母中的优化表达及活性鉴定  

Optimized Expression of Snake Fibrinolytic Enzyme Alfimeprase in Pichia pastoris and Its Activity Identification

作　　者：史婧[1] 张守涛[1] 齐亚飞[1] 郭蔼光[1] 

机构地区：[1]西北农林科技大学陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《中国生物工程杂志》2007年第5期28-33,共6页China Biotechnology

基　　金：陕西省农业分子生物学重点实验室创新基金

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,将其克隆至毕赤酵母分泌型表达载体pPICZαA,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pPICZαA-alf/GS115,且在优化条件下rALF的最高产量达425mg/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24kDa,且能与一抗特异结合。纤维蛋白平板法纤溶活性鉴定结果说明rALF获得了高活性分泌表达,即确立了一种优化表达体系,为ALF的深入研究和工业化生产应用奠定了重要基础。Alfimeprase （ALF） is a recombinantly modified variant of non-hemorrhagic zinc metalloproteinase fibrolase. The target gene alf was obtained from the clone vector p43-alf and cloned into the Pichia pastoris expression vector pPICZα A. Through high efficiency transformation and Zeocin selection, the recombinant strains of pPICZαA-alf/ GS115 were isolated. In order to achieve a high level expression of recombinant Alfimeprase （rALF）, optimization of pH value, methanol daily addition concentration, cell density and methanol induction time points were carried out, and the production of rALF reached up to 425 mg/L. By His · Bind chromatography, the purity of secreted rALF was as high as 95 %. SDS-PAGE and Westem blot analysis show that rALF has a molecular weight of about 24 kDa and is bound specifically to anti-His · tag monoclonal antibody. Activity identification results of the modified fibrin plate method demonstrate that the secreted rALF has high fibrinolytic activity. Thus sets up an optimized expression system for ALF, which will play an important role in its further studies and industrial production.

关 键 词：毕赤酵母 ALF rALF 纤维蛋白平板法 

分 类 号：Q786[生物学—分子生物学]