Construction of yeast Pichia pastoris to produce Man5GlcNAc2 mammalian mannose-type glycoprotein.
- Author:
Xiaopeng YANG
1
;
Bo LIU
;
Miao SONG
;
Xin GONG
;
Shaohong CHANG
;
Kuijing XUE
;
Jun WU
Author Information
1. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Gene Transfer Techniques;
Genetic Vectors;
genetics;
Glycoproteins;
biosynthesis;
Glycosylation;
Humans;
Mannose;
biosynthesis;
Oligosaccharides;
biosynthesis;
genetics;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
alpha-Mannosidase;
genetics
- From:
Chinese Journal of Biotechnology
2011;27(1):108-117
- CountryChina
- Language:Chinese
-
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.
