title: Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology.
abstract: Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.
authors.affiliation: Unit for Medical Biotechnology, Medical Biotechnology Center, VIB-UGent, Ghent, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: Laukens
authors.forename: Charlot
authors.initials: C
authors.lastname: De Wachter
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
mesh_terms: D004721:Endoplasmic Reticulum / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics; D006031:Glycosylation; D006056:Golgi Apparatus / Q000235:genetics; D006801:Humans; D008358:Mannose / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011134:Polysaccharides / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D008358:Mannose