重组人纤溶酶原基因的酵母表达、产物纯化及鉴定

目的: 研究重组人纤溶酶原丝氨酸蛋白酶结构域（rhPLG-SP）的酵母表达、纯化及理化性质。 方法:采用7.5 L发酵罐对巴斯德毕赤酵母(Pichia pastoris)工程菌 rhPLG-SP/GS115 进行高密度培养、甲醇诱导表达rhPLG-SP，培养液经三步纯化：超滤、Sephacryl S-100、SP-Sepharose FF，将活性组分透析后冷冻干燥。等点聚焦电泳、HPLC、质谱分别检测 rhPLG-SP等电点、纯度和分子量；纤维蛋白平板、肽底物S-2403 分别测定 rhPLG-SP激活后的纤维蛋白溶解和酰胺水解活性。结果: 7.5 L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rhPLG-SP纯度大于96%。理化分析显示 rhPLG-SP的等电点为7.5~7.8，分子量：27 877 Da，比活性：23.6U/mg。结论: 初步建立了rhPLG-SP酵母工程菌的高密度培养、表达及纯化工艺，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,Production Purification and Identification of Recombinant Human Plasminogen Gene in Yeast

Objective: To study the expression, purification and physicochemical characters of serine protease domain of human plasminogen（rhPLG-SP）in yeast. Methods: A 7.5L fermenter was used for high density culture of engineered Pichia pastoris rhPLG-SP/GS115 and methanol induction was performed to express rhPLG-SP. Fermentation broth was treated using a three-stage process: ultrafiltration, Sephacryl S-100 and SP-Sepharose FF. The active fraction was dialyzed and lyophilized. The isoelectric point (IP), purity, and molecular weight (MW) of rhPLG-SP were detected by IFE, HPLC and LC-MS respectively. Its fibrinolytic activity and amidolytic activity after activation were measured with fibrin plate and peptide substrate S-2403 respectively. Results: Fermentation in 7.5 L scale yielded an expression level of approximately 400mg rhPLG-SP per liter fermentation broth. Through this three-step purification procedure, the purity of rhPLG-SP could reach above 96%. The physicochemical analysis showed that the IP and MW of rhPLG-SP was 7.5~7.8 and 27 877 Dalton respectively, its specific activity was 23.6 U/mg. Conclusion: A pilot technologies for high density culture, expression and purification of engineered yeast containing rhPLG-SP gene had been set up. The activity of intermediate production was comparable to that of plasminogen extracted from human plasma, indicating a good perspective in scaling up production and application.