人微小纤溶酶原活性中心丝氨酸突变体基因在毕赤酵母中的表达

Expression of Serine Mutant of Microplasminogen at Active Site in Methylotrophic Yeast Pichia pastoris

用PCR方法将编码人微小纤溶酶原 (microplasminogen ,mPlg)活性中心Ser的密码子置换为Ala密码子 ,突变体cDNA与分泌型酵母表达载体 pHIL D8重组 ,构成受醇氧化酶基因 (AOX1)的启动子与转录终止区控制的酵母表达质粒 ,转化GS115酵母菌 ,经表型筛选、PCR扩增筛选阳性克隆 ,以甲醇诱导表达 ,Mm Plg分泌至培养液 ,经Westernblot证实。培养液经离心 ,上清与重组葡激酶 (Staphylokinase,Sak)孵育后 ,以酪蛋白 琼脂糖平板溶圈法、发色底物法检测 ,MmPlg未显示纤溶活性。

Recombinant microplasminogen with the active site Ser replaced by alanine was obtained by PCR based mutagenesis. Expression plasmid pMMLG was constructed by inserting of Mmplg cDNA into yeast expression vector pHIL D8 at BamHI and EcoRI sites, then transformed into GS115 cells. Positive clones were selected with MD/MM plates and confirmed by PCR. Several clones were incubated in BMG media and induced by 0.5% methanol in BMM media. The expression product Mmplg was secreted into medium. 32kD band appeared in SDS PAGE. The immunogenicity of the mplg was confirmed by Western blot. No fibrinolytic activity was shown by zymography and chromogenic substrate assay. Purified Mmplg will be used to prepare Sak·mplg crystal.