Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology
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.
Key wordsBi-antennary N-glycans Complex N-glycans DSA-FACE GlycoSwitch® Hybrid N-glycans N-Glycosylation engineering Pichia pastoris
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.
Financial and Competing Interests Disclosure
Declaration: The authors are either inventors or share otherwise in proceeds of licensing of patents and patent applications in the reviewed field.
- 13.Cipollo JF, Trimble RB (2002) The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man-residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus. Glycobiology 12:749–762PubMedCrossRefGoogle Scholar