Springer Protocols: Abstract: Modification of the N-Glycosylation Pathway to Produce Homogeneous, Human-Like Glycans Using GlycoSwitch Plasmids

Modification of the N-Glycosylation Pathway to Produce Homogeneous, Human-Like Glycans Using GlycoSwitch Plasmids

By: Wouter Vervecken², Nico Callewaert³, Vladimir Kaigorodov², Steven Geysens², Roland Contreras²

Abstract

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Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.

Affiliation(s): (2) Fundamental and Applied Molecular Biology, Department of Molecular Biomedical Research, Ghent University, Ghent, Belgium
(3) Department of Biology, Zürich, Switzerland

Book Title: Pichia Protocols

Series: Methods in Molecular Biology | Volume: 389 | Pub. Date: Aug-08-2007 | Page Range: 119-138 | DOI: 10.1007/978-1-59745-456-8_9

Subject: Biotechnology

Key Words: Pichia pastoris - glycosylation - N-glycans - glyco-engineering - GlycoSwitch - OCH1 - α-1,2-mannosidase - GlcNAc-transferase I - DSA-FACE - protein production - glycan analysis

References

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