人纤溶蛋白酶原K5在毕赤酵母中的表达及其生物活性鉴定  

Expression of rpK5 in Pichia pastoris and its Biological Activity Identification

作　　者：沈利[1] 宋大新[1] 高卜渝[1] 吕红[1] 李育阳[1] 

机构地区：[1]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海200433

出　　处：《生物工程学报》2004年第6期879-883,共5页Chinese Journal of Biotechnology

摘　　要：化学合成人纤溶蛋白酶原K5 (pK5 )的编码基因并克隆到毕赤氏酵母表达系统的分泌型载体pPIC9K上 ,将重组质粒经BglⅡ单酶切后电转化PichiapastorisGS115菌株 ,筛选出对G4 18有高抗性和在MM培养基上生长缓慢的转化子。经摇瓶发酵和甲醇诱导后 ,用 15 %SDS PAGE检测发酵上清液 ,表明有重组蛋白pK5的高表达。经CM Sepherose离子交换柱和Superdex 75分子筛层析两步分离纯化 ,获得了纯度达到 98%的rpK5。用MTT方法检测的结果表明 。The expression vector was constructed by inserting chemically synthesized DNA fragment coding for pK5 (plasminogen kringle 5) in expression vector pPIC9k and transformed into Pichia pastoris GS115. The transformants with high level of G418 resistance and slow growth on the MM plates were selected. After the selected transformant was grown in BMGY medium and induced by methanol, the culture supernatant was collected by centrifugation and analyzed by SDS-PAGE. The results showed that the recombinant pK5 was secreted into supernatant at quite high level. Through a simple two-step purification procedure, rpK5 with 98 % of purity was obtained. The results of endothelial cell proliferation assay showed that the in vitro anti-endothelial cell proliferation activity of purified rpK5 was significant.

关 键 词：人纤溶蛋白酶原K5 酵母 分泌表达 内皮细胞增殖分析 

分 类 号：Q786[生物学—分子生物学]