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Applied and Environmental Microbiology, May 2004, p. 2639-2646, Vol. 70, No. 5
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.5.2639-2646.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris
Wouter Vervecken,
Vladimir Kaigorodov, Nico Callewaert,,
Steven Geysens, Kristof De Vusser, and Roland Contreras*
Fundamental and Applied Molecular Biology, Department of Molecular Biomedical Research, Ghent University and Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium
Received 18 November 2003/ Accepted 29 January 2004
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 
-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and ß-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.
* Corresponding author. Mailing address: Fundamental and Applied Molecular Biology, Technologiepark 927, B-9052 Ghent-Zwijnaarde, Belgium. Phone: 32 9 331 3631. Fax: 32 9 331 3502. E-mail: Roland.Contreras{at}dmbr.UGent.be.
W.V., V.K., and N.C. contributed equally to this work.
Present address: Institute of Microbiology, Department of Biology, ETH Zürich, CH-8092 Zürich, Switzerland.
Applied and Environmental Microbiology, May 2004, p. 2639-2646, Vol. 70, No. 5
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.5.2639-2646.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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