title: Modification of the N-glycosylation pathway to produce homogeneous, human-like glycans using GlycoSwitch plasmids.
abstract: 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.
authors.affiliation: Department of Molecular Biomedical Research, Ghent University, Ghent, Belgium.
authors.forename: Wouter
authors.initials: W
authors.lastname: Vervecken
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kaigorodov
authors.forename: Steven
authors.initials: S
authors.lastname: Geysens
authors.forename: Roland
authors.initials: R
authors.lastname: Contreras
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D008361:Mannosidases / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D010843:Pichia / Q000201:enzymology; D010957:Plasmids / Q000235:genetics*; D011134:Polysaccharides / Q000096:biosynthesis*
chemical_list: D011134:Polysaccharides; D008364:Mannosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D008361:Mannosidases