A Pichia pastoris with α-1,6-mannosyltransferases Deletion and Its Use in the Expression of HSA/GM-CSF Chimera

Abstract

Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated. 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. Hence, a highly 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 into 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. Then the och1 deletion strain was applied to express a human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. Different from the hyperglycosylated HSA/GM-CSF chimera expressed in wild-type P. pastoris, the chimera expressed in the och1 deletion strain contained smaller N-glycan. The results suggested that the och1 mutant yeast may be more suitable for the production of recombinant glycoproteins. And the och1 deletion strain could be used for further re-engineering to produce complex human glycoproteins.