High-level expression of the mature region of human bone morphogenetic protein-4 in escherichia coli and the measurement of activity

Objective To further study the structure, cartilage and bone inductive mechanism and clinical application of human bone morphogenetic protein 4 (hBMP4). Methods The cDNA coding for the mature BMP4 was amplified by using reverse tranxdription polymerase chain reaction(RT PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P R P L promoter of a high level expression vector pBV220. Five positive recombinants (designated pBV/BMP4) were obtained by random selection, restriction digestion and SDS PAGE. hBMP4 was expressed in escherichia coli in a non fused form. Results SDS PAGE showed that a specific protein with a molecular weight of 14 at a level of 15.6% of the total bacterial proteins appeared in bacteria harboring pBV/BMP4, while this protein was absent in the control bacteria harboring pBV220. Sequence analysis of the amplified PCR products confirmed that the mature BMP4 was successfully cloned and expressed. hBMP4 stimulated alkaline phosphatase activity in a dose dependent manner. Conclusion The expressed product of hBMP4 in Escherichia coli is functionally active as demonstrated by the induction of alkaline phosphatase activity.