کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
35151 | 45078 | 2012 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli](/preview/png/35151.png)
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.
► RhBMP-2 homodimer was refolded effectively by simple dilution with yield 70%.
► A novel two-step DMF-aided hydrophobic interaction chromatography method was adopted.
► The active rhBMP-2 homodimer was separated from refolding mixture to homogeneity.
► The purification process was easy to scale up in industrial application.
Journal: Process Biochemistry - Volume 47, Issue 6, June 2012, Pages 960–967