Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli

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• 作者：Wangming Guo^a ; ^b ; Xiangcheng Zhu^a ; Jin Cai^a ; Lei Huang^a ; Peilin Cen^a ; Zhinan Xu^a ; ^{znxu@zju.edu.cn}
• 关键词：BMP ; bone morphogenetic protein ; TGF ; transforming growth factor ; rhBMP-2 ; recombinant human bone morphogenetic protein-2 ; rhBMP-7 ; recombinant human bone morphogenetic protein-7 ; HIC ; hydrophobic interaction chromatography ; DMF ; N ; N-dimethylformamide ; IB
• 刊名：Process Biochemistry
• 出版年：2012
• 期刊代码：29_13595113
• 类别：bio
• 出版时间：June, 2012
• 卷：47
• 期：6
• 页码：960-967
• 文件大小：862 K

摘要

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.