A combined system to engineer glycosylation efficiency and glycan structure in Saccharomyces cerevisiae.

We describe a novel synthetic N-glycosylation pathway to produce recombinant proteins carrying human-like N-glycans in Saccharomyces cerevisiae addressing at the same time glycoform and glycosylation efficiency. A double mutant strain Δalg3Δalg11, in which the N-glycans are not matured to their native high mannose structure, was used. In this mutant strain ...
lipid-linked Man(3)GlcNAc(2) is built up on the cytoplasmic side of the ER, flipped by an artificial flippase into the ER lumen, and then transferred with a high efficiency to the nascent polypeptide by a protozoan oligosaccharyltransferase. Protein bound Man(3)GlcNAc(2) serves directly as a substrate for Golgi targeted human N-acetylglucosaminyltransferases I and II. Our results confirmed the presence of the complex human-like N-glycan structure GlcNAc(2)Man(3)GlcNAc(2) on the secreted monoclonal antibody HyHEL-10. However, due to the interference of Golgi localized mannosyltransferases, heterogeneity of N-linked glycans was observed.
Appl. Environ. Microbiol.
Date: Nov. 30, 2012
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