Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system.
10.5051/jkape.2008.38.1.41
- Author:
Kyung Hee CHOI
1
;
Keumok MOON
;
Soo Hong KIM
;
Jeong Ho YUN
;
Kyung Lib JANG
;
Kyoo Sung CHO
Author Information
1. Research Development Institute, Cowellmedi Co. LTD., Korea.
- Publication Type:Original Article
- Keywords:
E.coli;
rhBMP-2;
purification;
alkaline phosphatase
- MeSH:
Acetonitriles;
Alkaline Phosphatase;
Blotting, Western;
Bone Morphogenetic Protein 2;
Chromatography, Affinity;
Chromatography, High Pressure Liquid;
Durapatite;
Electrophoresis, Polyacrylamide Gel;
Genetic Engineering;
Heparin;
Humans;
Molecular Weight;
Osteoblasts;
Recombinant Proteins;
Sprains and Strains;
Transforming Growth Factor beta
- From:The Journal of the Korean Academy of Periodontology
2008;38(1):41-50
- CountryRepublic of Korea
- Language:Korean
-
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.
