葡激酶在毕赤酵母中表达及纯化.docVIP

葡激酶在毕赤酵母中表达及纯化【摘要】 目的 探索葡激酶基因在毕赤酵母中的表达和纯化方法。方法 根据毕赤酵母的偏性合成了葡激酶基因，克隆到分泌型酵母表达载体pPIC9K中，重组载体线性化后，转化毕赤酵母GS115，甲醇诱导表达。应用 DEAE-Sepharose FF、Q-Sepharose FF和Sephacry1 S-200凝胶过滤层析法纯化表达产物，采用溶圈法对纯化产物进行生物学活性测定。结果 葡激酶在毕赤酵母中GS115的表达量约站总蛋白的45%；表达产物经DEAE-Sepharose FF、Q-Sepharose FF和SephacrylS-200纯化后纯度达到95%；测得其比活为5．2×10?4AU/mg。结论 利用毕赤酵母成功进行了重组葡激酶基因的表达及其表达产物的纯化。? 【关键词】葡激酶；毕赤酵母；表达和纯化 Expression and purification of staphylokinase in Pichia pastoris SONG Lei，LIU Hong-jun，LIU ning.Shandong Quangang Pharmaceutical Limited Company,Jinan,250014 ? 【Abstract】 Objective To study the expression staphylokinase gene in Pichia pastoris and the purification of expression product .Methods An artificial gene for staphylokinase was synthesized by using favored codons of the yeast Pichia pastoris.The gene was cloned into the secretory expression vector pPIC9K,and the recombinant vector was linearized and transformed into GS115,transformants had expression after inducement with methanol.The recombinant protein was purified through the steps of DEAE-Sepharose FF、Q-Sepharose FF和Sephacry1 S-200,and bioactivity was analyzed.Results The expression of staphylokinase in Pichia pastoris came up to 45%of total proteins;The recombinant protein was further purified by DEAE-Sepharose FF、Q-Sepharose FF和Sephacry1 S-200 to the 95%purity;purified was more than 5．2×10?4AU/mg.Conclusion Staphylokinasehas been expressed in Pichia pastoris and purified successfully? 【Key words】Staphylokinase;Pichia pastoris；Expression and purification 葡激酶(SAK)是金黄色葡萄球菌产生的一种胞外激酶，能特异地激活血浆纤溶酶系统溶解血栓。在此作用过程中，血浆纤维蛋白原浓度几乎不受影响，加之分子量小，有较强地穿透性，对富含血小板地血栓，尤其是脑动脉血栓和肢体静脉血栓地溶解较目前使用的链激酶和组织型纤溶酶原激活剂，具有溶栓速度快、毒副反应小和成本较低等特点，因而临床应用前景十分广阔[1-2]。目前，大肠杆菌表达的葡激酶已在国内上市，但存在产量低、不易纯化等问题。笔者使用的毕赤酵母是一种新型外源基因表达系统，具有许多明显优点，其诱导分泌表达的启动子AOX1可用甲醇严格调控，表达产物分泌到上清，不需要复杂的破菌手段。而且表达上清的杂蛋白含量很低，有利于进一步的分离纯化，是理想的表达系统[3]。 1 材料和方法 1．1 基因、菌种、载体 葡激酶基因由大连宝生物合成；大肠杆菌DH5α为本单位保存；酵母穿梭表达载体pPIC9K，Pichia Pastoris表达菌GS115(His-mut+),购自Invitrogen公司。 1．2 酶及其他试剂 限制性内切酶、T4 DNA连接酶、