The Expression Of HBmp-4 In The Pichia Pastoris And Its Site-Directed Mutagenesis

Bone morphogenetic proteins (BMPs) are growth factors that involved in the growth, differentiation and repair of a wide variety of tissues. They have been demonstrated to induce heterotopic bone formation and become increasingly recognized as valuable molecular tools for regenerating bone and accelerating fracture healing. In this study we have establish techniques to express recombinant human bone morphogenetic proteins 4 in the Pichia pastoris expression system. The cDNA mature domain that encodes hBmp-4 was cloned into the vector pPIC9K and the resulting construct was introduced into Pichia pastoris for expression. It was found with quantity dot blot that BMP-4 was expressed with a yield of about 29.82 mg/ml in shaken flask expression and accounts for 11.9% of the total protein in the supernatant. The expressed BMP-4 was further identified to be a 43,000 dalton molecular band with anti-hBMP4 antibody in western blot analysis. For more efficient expression of hBmp-4 in the Pichia pastoris, site-directed mutagenesis was performed to reconstruct hBmp-4 expression domain. Two Arg codon , CGC, with "0" codon usage were mutated to AGA with 86.6% codon usage, the favorite codon for Pichia pastoris.