Study On The Construction Of α-1,6-mannosyltransferases Gene Defects In Pichia Pastoris Strain

Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and were usually hyperglycosylated. This study is indispensable to the yeast N-glycosylation engineering project.α-1,6-mannosyltransferases gene (och1) encodes the enzyme that initiate the first step of out-chain elongation of high mannose type N-glycan in yeast, which is different in humans. So, deleting the initiating och1 gene could block the pathway to perform the hyperglycosylation and eliminate nonhuman glycosylation.At first, a high efficient method to knockout target gene by two-step recombination was established and was used to delete och1. In the first recombinant, a plasmid with och1::ADE1 and ura3 gene was linearized in the downstream of och1 and inserted to the och1 site of P.pastoris genome, where the upstream and downstream of och1 were duplicated. In the second recombinant, the duplicated fragments of och1 were exchanged and the och1 deletion strains were selected on the plates containing 5-FOA, but no adenine. The och1 deletion strain was constructed successfully and named GJK01.Then the yeast GJK01 was applied to express a human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. GM-CSF is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. A fusion protein consisting of GM-CSF and HSA has been generated to prolong its half-life. Different with the hyperglycosylated HSA/GM-CSF chimera expressed in wild type P.pastoris, the chimera expressed in yeast GJK01 contained smaller N-glycan.The results suggested that yeast GJK01 may be more suitable for production of recombinant glycoproteins. And it could be used for further re-engineering to produce complex human glycoproteins.