Use of HDEL‐tagged Trichoderma reesei mannosyl oligosaccharide 1,2‐α‐D‐mannosidase for N‐glycan engineering in Pichia pastoris

FEBS Letters

Contreras, Roland²  Callewaert, Nico²  Min Jou, Willy¹  Saelens, Xavier¹  Geysens, Steven²  Laroy, Wouter²  Cadirgi, Hasan² 

[1] Unit of Molecular Virology, Department of Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, K.L.-Ledeganckstraat 35, B-9000 Ghent, Belgium;Unit of Fundamental and Applied Molecular Biology, Department of Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, K.L.-Ledeganckstraat 35, B-9000 Ghent, Belgium

关键词: Yeast;    Glycosylation;    1;    2-α-Mannosidase;    Endoplasmic reticulum;    Retention signal;    Pichia pastoris;    Man;    mannose;    GlcNAc;    N-acetylglucosamine;    N-glycan;    asparagine-linked glycan;    PCR;    polymerase chain reaction;    SDS–PAGE;    sodium dodecyl sulphate–polyacrylamide gel electrophoresis;    GAP;    glyceraldehyde-3-phosphate dehydrogenase;    AOXI;    alchohol oxidase I;   

DOI  :  10.1016/S0014-5793(01)02676-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-α-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of α-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man₅GlcNAc₂ was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.

【 授权许可】

Unknown