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Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system  

Choi, Kyung-Hee (Research Development Institute, Cowellmedi Co. LTD.)
Moon, Keumok (Research Development Institute, Cowellmedi Co. LTD.)
Kim, Soo-Hong (Research Development Institute, Cowellmedi Co. LTD.)
Yun, Jeong-Ho (Department of Dentistry, College of Medicine, Kwandong University, Myongji Hospital)
Jang, Kyung-Lib (Depatment of Biological Science, College of Natural science, Pusan National University)
Cho, Kyoo-Sung (Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University)
Publication Information
Journal of Periodontal and Implant Science / v.38, no.1, 2008 , pp. 41-50 More about this Journal
Abstract
Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.
Keywords
E. coli; rhBMP-2; purification; alkaline phosphatase;
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