title: [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 alpha-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(delta185)] or Arabidopsis thaliana [ATMDSI(delta48)], 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(delta48) 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.
authors.affiliation: Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Yang
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.forename: Miao
authors.initials: M
authors.lastname: Song
authors.forename: Xin
authors.initials: X
authors.lastname: Gong
authors.forename: Shaohong
authors.initials: S
authors.lastname: Chang
authors.forename: Kuijing
authors.initials: K
authors.lastname: Xue
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
mesh_terms: D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000096:biosynthesis; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D043323:alpha-Mannosidase / Q000235:genetics*
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C058642:mannosyl(5)-N-acetyl(2)-glucose; D043323:alpha-Mannosidase; D008358:Mannose