Cloning And Expression Of Encoding Sequence Of Mature Peptide For Bone Morphogenetic Protein-7

Bone morphogenetic protein-7(BMP-7), also known as osteogenetic protein-1 (OP-1), belongs to the TGF- B superfamily. Besides the induction of bone and cartilage formation by promoting osteoblastic and chondroblastic differentiation, with the development of study about other effects and signal transduction , BMP-7 may have broader effects in vivo than hitherto recognized. Its cDNA encodes a 431 amino acid propeptide, of which the 29 amino acid signal peptide and the 263 amino acid prodomin are cleaved, leaving a 139 amino acid mature peptide. In vivo, two molecules homodimerize to exert its bioactivities.In the present study, we amplified the DNA sequence encoding for mature peptide of BMP-7, using pBV220/BMP-7 as the template. The amplified sequence was inserted into pQE30. The procedures are described as follows: The amplified sequence, which had BamH I Pst I sites at 5' and 3'ends,respectively, was connected to the vector pQE30. Then the recombinant plasmid was transformed into E.coli M15 [pREP4] for expression. The molecular weight of 6xHis-BMP-7 is about 16.78kD. This fusion protein existed mainly in the form of insoluble inclusion bodies. It could be recognized through Western blotting, by the polyclonal antibody against hBMP-7 from goat. After optimization of conditions, the fusion protein accounted for 41.95% of the total cellular proteins. The inclusion bodies were dissolved, followed by purification using one-step Ni-NTA agrose affinity-chromatography under denaturing conditions. After that, the purified protein was refolded through gradient dialysis. The yiled of the recombinant protein was 58.8mg/L medium. Finally, it was confirmed that the recombinant protein was biologically active, which induced the activity of Alkine phosphatase (ALP) in cell line of MG63.