| Bone morphogenetic proteins ( BMPs ) are growth factors involved in the growth, differentiation and repair of a wide variety of tissues. They have been demonstrated to induce ectopic bone formation and become increasingly recognized as valuable molecular tools for regenerating bone and accelerating fracture healing. In this study, the recombinant human bone morphogenetic proteins-7( rhBMP-7 ) was sussessfully expressed in the Pichia pastoris expression system. Furthermore, investigation for rhBMP-7 expression, small scale fermentation and activities has been carried out. The cDNA mature domain that encodes hBMP-7 was cloned into the vector pPIC9K and the resulting construct was introduced into Pichia pastoris strain KM71 for expression. For higher expression level, site-directed mutagenesis was performed to reconstruct hBMP-7 expression domain. Three Arg codon , CGG,CGA,CGG, with "0" codon usage in yeast were mutated to AGA with 86.6% codon usage, the favorite codon for Pichia pastoris. Tricine-PAGE and Western-Blotting analysis of culture medium indicated that the molecular weight of rhBMP-7 expressed in Pichia pastoris is about 18KD, with a yield of about 25. 45 ug/ml in shaken flask, accounts for 76.2% of the total protein in the supernatant. The reconstructed expression domain with favorite codon for Pichia pastoris. show a remarkably increased level of expression with 4~5-fold more potent than the originone. Observation of several inducing condition showed that expression level reach highest at about day 5 after induction, the optimal concentration of methanol induction is 1%, and the optimal OD600 before induction is about 10. Primary activity of rhBMP7 was investigated by the inductin of ectopic cartilage and bone in rat. The rhBMP7 was lyophilized onto type I collagen, and then implanted into the leg muscle of male Long-Evans rats. The result show that rhBMP7 can stimulate the formation of cartilage in rats on day 10 after implant.
|
PDF Full Text Request