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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
The PAI-1 cDNA encoding 379 amino acid residues was inserted into the methanolic yeast vector containing the AOX1 promoter and PHO1 secretion signal peptide sequence to construct the expression plasmid pYIS-1. Expression Plasmid Transformation P. pastoris methanol-containing yeast cells, His + Mut-phenotype transformants were screened. After being cultured in low density shake flask and induced with 1% methanol for 7 days, the culture supernatants were confirmed by SDS-PAGE, PAI-1 bioassay and Western blot Four PAI-1 bands with a molecular weight of 43-51 kD. The expressed PAI-1 was effectively secreted into the culture medium, accounting for about 26% of the total protein in the culture medium, up to 4 mg / L; its specific activity was 1.16 × 104 AU / mg. Different molecular weight PAI-1 may be related to the degree of glycosylation