High Level Expression And Dimeric Research Of HBMP-7 In Yeast Pichia Pastoris

Bone morphogenetic protein-7 (BMP-7) is a key signaling molecule involved inmultiple organogenesis and development, and has bone or cartilage inductiveactivities.The methylotrophic yeast Pichia pastoris has become a highly populareukaryotic expression host system for the recombinant production of a wide variety ofproteins. Here, we report:(1) Increase hBMP-7 protein production in Pichia pastoris with multiple copies numberof synthetic genes of hBMP-7 mature domain, fused to the S. cerevisiae pre-proalpha-factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1)promoter and terminator sequences. However, hBMP-7 protein production is monomer.(2) Replace the amino acid sequence -RSIR- between the hBMP-7 prodomain and maturedomain into -KR- by SOEPCR, then clone the mutant DNA into pPIC9K, construct theexpression plasmid: pPIC9K-hBMP-7-pmk. Analysis of the expression of thehBMP-7-pmk in Pichia pastoris, but there are no hBMP-7 homodimers.(3) Clone hBMP-7 prodomain and mature domain cDNA into pPIC9K, and clone furincDNA into pAO815, to construct plasmids" pPIC9K-hBMP-7-pm and pAO815-furin.Then transform Pichia pastoris with the plasmids: pPIC9K-hBMP-7-pm and pAO815-furin. Screen the transformation of Pichia pastoris that have hBMP-7-pm and furin.