Pichia pastoris X-33 with OCH1 gene deletion and its expression of glycoprotein GM-CSF

Abstract:

Abstract: [Objective] Glycoproteins derived from yeast expression systems are usually hyperglycosylated and contain non human N-glycans of the high mannose type. α-1,6-mannosyltransfe rase(och1p) plays a key role in modifying glycoproteins with high mannose-type N-glycans. Therefore, we engineered a P. pastoris X-33 strain with OCH1 gene deletion. This strain was further used as a host for production of glycoproteins with smaller N-glycans. [Methods] First, we knockout the URA3 gene of P.pastoris X-33 which encoding orotidine-5’-phosphate decarboxylase by double homologous recombination. Then, we knockout OCH1 gene using URA3 gene as a selecting marker, and obtained X-33( och1-) strain. After that, this mutant strain was used to expression the glycoprotein granulocyte-macrophage colony-stimulating factor (GM-CSF). [Results] Different from hyperglycosylated GM-CSF expressed in wild type P.pastori X-33, the glycoprotein expressed in X-33(och1-), containing smaller N-glycan. [Conclusion] The results suggested that X-33(och1-) strain can be used as an expression host for production of glycoproteins lacking the outer-chain hypermannoses, and a host could be used for further N-glycosylation engineering.

Key words:Keywords: N-glycosylation, Pichia pastoris X-33, Gene knockout, Granulocyte-macrophage colony-stimulating factor（GM-CSF）