Construction of yeast Pichia pastoris to produce Man5GlcNAc2 mammalian mannose-type glycoprotein

Abstract:

Glycosylation is vital for activity, higher structure and function of protein. Glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated, while those from mammalian cells contain N-glycan of hybrid or complex type. We introduced the α-1,2-mannosidase I (MDSI) into yeast cells, which catalyzed an essential proceeding of N-glycan structures from Man₈GlcNAc₂ to Man₅GlcNAc₂. The plasmids contained MDSI genes from Homo sapiens [HMDSI(Δ185)] or Arabidopsis thaliana [ATMDSI(Δ48)], and three ER-signals were used to be transformed a mutant Pichia pastoris GJK01 , respectively. The reporter protein HSA/GM-CSF (human serum albumin and granulocyte-macrophage colony stimulating factor fusion protein) was expressed and its N-glycans were analyzed by DSA-FACE (DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis). The plasmid contained ER-ScMnsI-ATMDSI(Δ48) was expressed in Pichia pastoris, the Man₅GlcNAc₂ N-glycan on secreted glycoprotein HSA/GM-CSF was observed. The research reported here provided basic substrate to obtain the hybrid- and complex-type glycans in mammalian cell.