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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Acknowledgements
B.L. and C.D.W. hold a fellowship of the Institute for the Advancement of Scientific and Technological Research in Industry (IWT). This work was in part funded by grant no. G.0.541.08.N.10 of FWO-Vlaanderen and an ERC Consolidator grant (GlycoTarget). We acknowledge Dr. D. Vanderschaeghe and Kurt Gehlsen for useful suggestions while preparing the manuscript and for carefully reading the manuscript.
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Laukens, B., De Wachter, C., Callewaert, N. (2015). Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology. In: Castilho, A. (eds) Glyco-Engineering. Methods in Molecular Biology, vol 1321. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2760-9_8
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DOI: https://doi.org/10.1007/978-1-4939-2760-9_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2759-3
Online ISBN: 978-1-4939-2760-9
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