Affiliation:
1. Fundamental and Applied Molecular Biology, Department of Molecular Biomedical Research, Ghent University and Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium
Abstract
ABSTRACT
The
Pichia pastoris
N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas
Pichia
cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the
P. pastoris
N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of
OCH1
and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and
N
-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any
P. pastoris
expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
161 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献