Soluble Expression and Purification of Bioactive Recombinant Human Bone Morphogenetic Protein-2 from Escherichia coli

Abstract

Human bone morphogenetic protein-2 (hBMP-2) is a potent growth and differentiation factor for bone induction and regeneration. Recombinant hBMP-2 (rhBMP-2) was cloned and expressed as a soluble protein using E.coli-based expression system. A full-length gene encoding mature hBMP-2was amplified by RT-PCR, cloned into an expression vector and expressedusing SHuffle E. coli cells. The rhBMP-2 was successfully expressed as asoluble protein under the control of the lacUV5 and protein A promoters byIPTG induction. The rhBMP-2 fused with ZZ domain at its N-terminus wassuccessively purified with a single step by using IgG Sepharose 6 fast flowaffinity chromatography. Analysis of the purified protein on SDS-PAGE,Western blot analysis and LC-MS/MS, verified that the purified protein wasrhBMP-2. The biological activity testing on hFOB 1.19 showed that rhBMP-2had the ability to significantly induce cell proliferation in a dose dependentmanner. ALP staining and activity assay also increased after rhBMP-2 treatment. The mRNA expression of the osteogenic genes by quantitative real-time PCR (qRT-PCR) showed that rhBMP-2 was able to up-regulate the gene expression of ALP, COL1, BMP-2, Runx2, and OPN. This data indicates that rhBMP-2 is functionally active to induce human osteoblast proliferation and differentiation. The production of rhBMP-2 by this developed method could be useful for bone regeneration and repair applications.