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Expression, Production Purification and Identification of Recombinant Human Plasminogen Gene in Yeast |
CHEN Wu1,MO Wei2,ZHANG Yan-lin2,SONG Gang2,SONG Hou-yan2 |
1.The Life Science and Biopharmacy College, Guangdong Pharmaceutics University, Guangzhou 510006, China
2.The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China |
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Abstract 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.
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Received: 23 June 2009
Published: 29 October 2009
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Corresponding Authors:
Chen Wu
E-mail: cwy_100@163.com
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