Ⅰ型纤溶酶原激活物抑制剂cDNA在P.pastoris酵母菌中的表达和鉴定

将编码３７９个氨基酸残基的ＰＡＩ－１ｃＤＮＡ插入到含ＡＯＸ１启动子和ＰＨＯ１分泌信号肽序列的甲醇营养型酵母载体中，构建成表达质粒ｐＹＩＳ－１．表达质粒转化Ｐ．ｐａｓｔｏｒｉｓ甲醇营养型酵母细胞，筛选Ｈｉｓ＋Ｍｕｔ－表型的转化子，经低密度摇瓶培养，１％甲醇诱导表达７ｄ后，培液经ＳＤＳ－ＰＡＧＥ分析，ＰＡＩ－１生物活性测定和Ｗｅｓｔｅｒｎｂｌｏｔ证实表达出分子量为４３～５１ｋＤ的４条ＰＡＩ－１条带．表达的ＰＡＩ－１能有效地分泌到培液中，占培液总蛋白的２６％左右，达４ｍｇ／Ｌ培液；其比活性为１．１６×１０４ＡＵ／ｍｇ．不同分子量ＰＡＩ－１可能与其糖基化程度不同有关

Expression and Characterization of Type 1 Plasminogen Activator Inhibitor in Pichia pastoris

By inserting PAI 1 cDNA coding 379 amino acid residues into the methylotrophic yeast expression vector pHIL S1 containing AOX1 promotor and the sequences of PHO1 secreting signal peptides,the expression plasmid pYIS 1 was constructed and then transformed into P.pastoris GS115.The His + Mut - transformants were selected and fermentated in flasks with low density.After 7 days of 1% methanol induction,the expression media were analyzed and the expressed protein was verified to be recombinant human PAI 1(rh PAI 1) in different molecular weights of 43—51 kD by SDS PAGE,bioactivity and Western blot detection.The expressed rhPAI 1 could be effectively secreted into media,and its highest quantity was estimated to be 26% of total proteins or 4 mg/L in media.The specific activity of rhPAI 1 was 1.16×10 4 AU/mg.The non homogeneity of M r of expressed rhPAI 1 may be related to the length of its oligosaccharide chains.The PAI 1 expressed in P.pastoris was very helpful to further study its structure and physiological function.