Expression, purification, and bone-inducing activity of recombinant human bone morphogenetic protein-3 mature peptide.

It was inferred that the mature peptide of human bone morphogenetic protein-3 (hBMP-3m) consists of the carboxyl terminal 127 amino acid residues of hBMP-3. A plasmid, pDH-B3m, was constructed by inserting the cDNA sequences encoding hBMP-3m into pDH, a PL-containing expression vector. pDH-B3m was transformed into Escherichia coli DH5 alpha. The highest expression level of recombinant hBMP-3m (rhBMP-3m) could be reached after 6 hours of induction at 42 degrees C, accounting for 28% of the total bacterial proteins. The rhBMP-3m was found in the inclusion bodies. After being washed and partially purified, the inclusion bodies were solubilized in urea and purified efficiently through ion-exchange chromatography. The purity of the rhBMP-3m was at least 95%. The rhBMP-3m was refolded by dilution method and then 1 mg was implanted into mouse thigh muscle to assay its activity. A classic pattern of cartilaginous osteogenesis was observed. The results showed that the purified and refolded rhBMP-3m had ectopic bone-inducing activity.