Chapter title |
Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology
|
---|---|
Chapter number | 8 |
Book title |
Glyco-Engineering
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2760-9_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2759-3, 978-1-4939-2760-9
|
Authors |
Laukens, Bram, De Wachter, Charlot, Callewaert, Nico, Bram Laukens, Charlot De Wachter, Nico Callewaert |
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. |
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