A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae

Farnoush Parsaie Nasab, Markus Aebi, Gesche Bernhard, Alexander Daniel Frey

Research output: Contribution to journalArticleScientificpeer-review

38 Citations (Scopus)

Abstract

We describe a novel synthetic N-glycosylation pathway to produce recombinant proteins carrying human-like N-glycans in Saccharomyces cerevisiae, at the same time addressing glycoform and glycosylation efficiency. The Δalg3 Δalg11 double mutant strain, in which the N-glycans are not matured to their native high-mannose structure, was used. In this mutant strain, lipid-linked Man3GlcNAc2 is built up on the cytoplasmic side of the endoplasmic reticulum, flipped by an artificial flippase into the ER lumen, and then transferred with high efficiency to the nascent polypeptide by a protozoan oligosaccharyltransferase. Protein-bound Man3GlcNAc2 serves directly as a substrate for Golgi apparatus-targeted human N-acetylglucosaminyltransferases I and II. Our results confirmed the presence of the complex human-like N-glycan structure GlcNAc2Man3GlcNAc2 on the secreted monoclonal antibody HyHEL-10. However, due to the interference of Golgi apparatus-localized mannosyltransferases, heterogeneity of N-linked glycans was observed.
Original languageEnglish
Pages (from-to)997-1007
Number of pages11
JournalApplied and Environmental Microbiology
Volume79
Issue number3
DOIs
Publication statusPublished - 2013
MoE publication typeA1 Journal article-refereed

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