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| High-level Expression and Purification of Yarrowia lipolytica Lipase Lip2 with Six Hisditine Tags in Pichia pastoris |
| WANG Xiao-feng, SHEN Xu-guang, ZHAO He-yun, SUN Yong-chuan, JI Chang-tao, YAN Yun-jun |
| Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science & Technology,Wuhan 430074,China |
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Abstract The mature Y. lipolytica lipase Lip2 gene without the signal sequence and with a 6×His-tag in N-terminal,was ligated into vector pPIC9K. The recombinant plasmid pPIC9K-Lip2H was linearized by rectriction enzyme Sal I,and then transformed into Pichia pastoris GS115 by electroporation. A clone exhibiting a maximum lipase activity of 400U/ml in shaking flask was chosen for 10 L bioreactor cultivation. The fed-batch fermentation process was preliminarily optimized.With respect to the effects on biomass and the expression level of His6-YlLip2,the optimal basal salt medium was FM22,and the optimal parameters for induction pH and temperature were 5.0,25℃,respectively. After 114h methanol induction,the lipase activity of His6-YlLip2 reached the highest value of 3160U/ml. SDS-PAGE analysis showed that the molecular weight of the aimed protein was about 38 kDa. Using a Ni-NTA affinity chromatography,the purity of recombinant His6-YlLip2 reached 95.43% by one-step purification with the specific activity of 4250U/mg.
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Received: 07 December 2010
Published: 26 April 2011
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