人纤溶酶原kringle区缺失突变体的毕赤酵母表达、产物纯化及鉴定

目的研究人纤溶酶原kringle区缺失突变体（PLGAK）的毕赤酵母（Pichia pastoris）表达、产物纯化及理化性质鉴定。方法采用7．5L发酵罐对工程菌PLG△K／GS115进行高密度培养、甲醇诱导表达，培液经离心、超滤、离子交换层析、凝胶滤过、透析后冷冻干燥。等电聚焦电泳、HPLC、质谱分别检测PLG△K等电点、纯度和分子量；纤维蛋白平板、肽底物S-2403分别测定PLG△K激活后的纤维蛋白和酰胺水解活性。结果7．5L高密度发酵可获得约为400mg／L培液的表达量，经三步纯化后制备的PLG△K纯度大于96％。理化分析显示PLG△K的等电点为7．5～7．8，分子量：27．787kD，比活性：23．6U／mg。结论初步建立了PLG△K的酵母高密度发酵及纯化工艺，所制备半成品活性与血浆提取的PLG相近，具备放大生产和应用的潜力。

Expression,purification and identification of kringle domain deletion mutant of human plasminogen in Pichia pastoris

Objective To investigate the expression, purification and characters of the kringle domain deletion mutant of human plasminogen （PLG△K） in Pichia pastoris. Methods Fermentation at high density （ A600 〉 200） in a 7.5 L fermenter was carried out and PLG△K was purified from the culture broth in a three step-process： ultrafiltration, gel filtration, ion exchange chromatography, and was lyophilized after dialysis. The IFE, HPLC and LC-MS were used to detect its isoelectric point （IP）, purity, and molecular weight （MW）. The fibrinolytic activity and amidolytic activity were measured with fibrin plate and chromogenic peptide substrate S-2403 respectively. Results Fermentation in 7. 5 L scale yielded an expression level of approximately 400 mg PLG△K per liter fermentation broth. Through three steps of purification, the PLG△K had a purity of over 96%. The exact MW of PLG△K was 27. 787 kD examined by LC-MS and its IP was 7.5 - 7.8. Conclusion A pilot expression and purification technology of PLG△K in Pichia pastoris cultured at high density has been set up. The activities of intermediate production are comparable to that of plasminogen extracted from human plasma, indicating a good perspective in scale up and application.