Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli |
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Authors: | Wangming Guo Xiangcheng Zhu Jin Cai Lei Huang Peilin Cen Zhinan Xu |
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Institution: | 1. Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China;2. Hangzhou Jiuyuan Gene Engineering Co., Ltd., Hangzhou, Zhejiang 310018, PR China |
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Abstract: | To renature the inactive rhBMP-2 which overexpressed in Escherichia coli, post-expression treatments including inclusion bodies solubilization and in vitro refolding were systematically investigated. An optimized refolding process was established from screening and successfully scaled up with yield greater than 70%. Then, hydrophobic interaction chromatography (HIC) was adopted as two consecutive stages to separate the active rhBMP-2 homodimer from refolding mixture. Aiding additive N,N-dimethylformamide (DMF) was found to enhance the resolution of rhBMP-2 homodimer most effectively. The rhBMP-2 homodimer was purified to homogeneity through two HIC separations at different salt contents, the purified rhBMP-2 homodimer was fully bioactive and had equivalent biological activity to rhBMP-2 produced from Chinese hamster ovary cell (CHO). Under the optimal refolding and purification conditions, 80 mg rhBMP-2 homodimer with high purity could be obtained from 1 g wet weight of inclusion bodies. Finally, this efficient refolding and purification procedure was successfully scaled up in the pilot pharmaceutical plant. |
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