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

Paper ID Volume ID Publish Year Pages File Format Full-Text
35151 45078 2012 8 PDF Available
Title
Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli
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.

► 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.

Keywords
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
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Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 47, Issue 6, June 2012, Pages 960–967
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us