Prodomain Of HBmp4 Expressed As Fusion Protein To Mature Domain In Pichia Pastoris & Its Effect On Fold Of HBmp4

Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor-p (TGF-P) superfamily, were originally identified as protein regulators of cartilage and bone formation. However, they have since been shown to be involved in embryogenesis and morphogenesis of various tissues and organs. BMPs have also been shown to regulate the growth, differentiation, chemotaxis and apoptosis of various cell types, including mesenchymal cells, epithelial cells, hematopoietic cells and neuronal cells.In recent years, the human BMP-4 has been produced in CHO cells and Escherichia coli by recombinant DNA methods and recognized as a potentially powerful clinical tool. Although recombinant BMP-4 produced in mammalian cells or E. coli can induce new bone formation, naturally occurring BMP-4 appeared to be more active than recombinant preparations. In vivo,BMP-4 is synthesized as an inactive precursor(preproprotein) that is cleaved after the multibasic motif to yield the active, carboxyl-termainal mature protein dimmer. So we surmise that propeptide perhaps act as intramolecular chaperone(IMC).In this study, The sequence of prodomain and mature domain that encodes hBMP-4 was cloned into the vector pPIC9K and the resulting construct was introduced into Pichia pastoris strain GS115 for expression. For propeptide efficiently cleave, site-directed mutagenesis was performed toreconstruct sequence Arg-Ala-Lys-Arg (R-A-K-R) to Lys-Arg-Lys-Arg(Kex2) that the Kex2P cleaves in Saccharomyces cerevisiae. Cultures in shaking bottle showed the optimum conditions as follows: the culture medium is BMGY/BMMY; the pH is 6.0; methanol concentration is 1%; inducement period is 96h. SDS-PAGE and Western-Blotting analysis of culture indicated that the molecular weight of rhBMP-4/pro-on expressed in Pichia pastoris is about 66KD, because the sequence Arg-Ala-Lys-Arg (R-A-K-R) didn't reconstruct to the sequence Kex2. And the molecular weight of ThBMP-4/pro-mut is about 22KD. But we didn't found homodimers in culture medium.