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Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology

Nature Protocols volume 4, pages 58–70 (2009)Cite this article

Abstract

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes ∼3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal2GlcNAc2Man3GlcNAc2N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

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Figure 1: Glycoengineering strategy overview.
Figure 2: SDS-PAGE analysis of medium proteins from mIL-10-producing strains.
Figure 3: DSA-FACE profiles for mIL-10.
Figure 4: DSA-FACE and SDS-PAGE analysis of medium proteins from mGM-CSF-producing strains.
Figure 5: DSA-FACE and SDS-PAGE analysis of medium proteins from mIL-22-producing strains.

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Acknowledgements

We thank Annelies Van Hecke for technical assistance and Dr. Bennet Cohen and Dr. David Bramhill for fruitful discussions. This work was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and Research Corporation Technologies (Tucson, AZ, USA).

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Authors and Affiliations

  1. Department for Molecular Biomedical Research, Unit for Molecular Glycobiology, VIB, Technologiepark 927, Ghent (Zwijnaarde), B-9052, Belgium

    Pieter P Jacobs, Steven Geysens, Wouter Vervecken, Roland Contreras & Nico Callewaert

  2. Department of Molecular Biology, Ghent University, Technologiepark 927, Ghent (Zwijnaarde), B-9052, Belgium

    Pieter P Jacobs, Steven Geysens, Wouter Vervecken & Roland Contreras

  3. Department of Biochemistry, Laboratory for Protein Biochemistry and Biomolecular Engineering, Physiology and Microbiology, Ghent University, K.L.-Ledeganckstraat 35, Ghent, B-9000, Belgium

    Nico Callewaert

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  1. Pieter P Jacobs
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Correspondence to Nico Callewaert.

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Jacobs, P., Geysens, S., Vervecken, W. et al. Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc 4, 58–70 (2009). https://doi.org/10.1038/nprot.2008.213

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