Pichia / Hgm-csf Engineering Bacteria And Expression Analysis

Construction and Assay of recombinant Pichia pastoris strainscontaining hGM-CSF geneYang GuoFeng(South China University of Tropical Agriculture, Haikou, Hainan 571101)Abstract The gene encoding human granulocyte-macrophage colony-stimulating factor(hGM-CSF) was cloned to a downstream site of promoter GAP of pGAPZ a ?A vector. The recombinant expression vector was transfected into SMDI 168 strain(his4, pep4) and GS 115 strain(his4) of Pichia pastoris. SDS-PAGE analysis of secretory proteins indicated that three recombinant strains( 1 ~? 2#, 6#) were able to express 14.454-15.l35kD proteins at high level whose relative molecular mass are corresponding to hGM-CSF. The supernatant of recombinant yeast cultures was assayed for hGM-CSF activity using MTT method. The results showed that these proteins expressed by recombinant strains had the same activity as natural hGMCSF and the specific activity were 1.043 X 107U/mg(1#),O.887 X l07U/mg(2~) and 1.034>( 1 07U/mg(6#). These data demonstrated the feasibility of production of recombinant hGM-CSF in Pichia pastoris initially.