中国生物工程杂志

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.