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KMID : 0363020080380010041
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Journal of Korean Academy of Periodontology
2008 Volume.38 No. 1 p.41 ~ p.49
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Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system
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Choi Kyung-Hee
Moon Keum-Ok
Kim Soo-Hong
Yun Jeong-Ho
Jang Kyung-Lib
Cho Kyoo-Sung
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Abstract
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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 & 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. (J Korean Acad Periodontol 2008;38:41-50)
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KEYWORD
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E.coli, rhBMP-2, purification, alkaline phosphatase
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