Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system
- Affiliations
-
- 1Research Development Institute, Cowellmedi Co. LTD., Korea.
- 2Department of Dentistry, College of Medicine, Kwandong University, Myongji Hospital, Korea.
- 3Depatment of Biological Science, College of Natural science, Pusan National University, Korea.
- 4Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Korea. kscho@yuhs.ac
- KMID: 1486783
- DOI: http://doi.org/10.5051/jkape.2008.38.1.41
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.
Keyword
MeSH Terms
-
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
Acetonitriles
Alkaline Phosphatase
Bone Morphogenetic Protein 2
Durapatite
Heparin
Recombinant Proteins
Transforming Growth Factor beta
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