Overexpression Of Recombinant Human Bone Morphogenetic Protein-2 In E.coli And In VItro Refolding

Bone morphogenetic protein-2 (BMP-2) is a member of the bone morphogenetic protein family (BMPs), which has been implicated to be involved in biological functions. BMP-2 was originally identified as the protein regulator of cartilage and bone formation. Indeed, it has been shown to be involved in embryogenesis and morphogenesis of various tissues and organs. Therefore, it is suggested that the potential is huge for hBMP-2 to benefit the treatment of a variety of diseases.Using the techniques of molecular cloning and DNA point mutation skill, the mature rhBMP-2 cDNA was inserted into the pET35b(+), which was transformed into the Eschericha coli strain BL21(DE3). The strain, called as JH, expressed the high-level rhBMP-2. The optimum condition was obtained after the recombinant E.coli strain JH culture conditions and expression character were studied. The rhBMP-2 was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. The denatured rhBMP-2 was obtained by DEAE chromatography. The reduced and denatured rhBMP-2 monomers were refolded and dimerized in the redox systems (reduced and oxidized glutathione) by means of a simple dilution method. Finally, a one-step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP-2 dimers and monomers. The purified rhBMP-2 dimers showed its biological activity by the induction of alkaline phosphatase activity produced by C2C12 cells. The in vitro refolding procedure of rhBMP-2 could apply to the TGF-P superfamily, because of their highly conservative structure Cys-knot. Furthermore, the highly active rhBMP-2 was obtained in this simple and effective in vitro refolding procedure, which could be avaible for the large-scale industrial production and play an important role in the biological and clinical research of rhBMP-2.