Abstract
Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides Annu. Rev. Biochem. 54, 631–664.
Lee, S. J., Evers, S., Roeder, D., et al. (2002) Mannose receptor-mediated regulation of serum glycoprotein homeostasis. Science 295 1898–1901.
Vervecken, W., Kaigorodov, V., Callewaert, N., Geysens, S., De Vusser, K., and Contreras, R. (2004) In vivo synthesis of mammalian like hybrid type N-glycans in Pichia pastoris. Appl. Environ. Microbiol. 70, 2639–2646.
Jackson, P. (1990) The use of polyacrylamide-gel electrophoresis for the highresolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem. J. 270, 705–713.
Maras, M., De Bruyn, A., Schraml, J., et al. (1997) Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RUTC 30. Eur. J. Biochem. 245, 617–625.
Martinet, W., Saelens, X., Deroo, T., et al. (1997) Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase. Eur. J. Biochem. 247, 332–338.
Maras, M., Callewaert, N., Piens, K., et al. (2000) Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-D-mannosidase. J. Biotechnol. 77, 255–263.
Callewaert, N., Laroy, W., Cadirgi, H., et al. (2001) Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for Nglycan engineering in Pichia pastoris. FEBS Lett. 503, 173–178.
Ballou, C. E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol. 185, 440–470.
Callewaert, N., Geysens, S., Molemans, F., and Contreras, R. (2001) Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNAsequencing equipment. Glycobiology 11, 275–281.
Papac, D. I., Briggs, J. B., Chin, E. T., and Jones, A. J. (1998) A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Glycobiology 8, 445–454.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Vervecken, W., Callewaert, N., Kaigorodov, V., Geysens, S., Contreras, R. (2007). Modification of the N-Glycosylation Pathway to Produce Homogeneous, Human-Like Glycans Using GlycoSwitch Plasmids. In: Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 389. Humana Press. https://doi.org/10.1007/978-1-59745-456-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-59745-456-8_9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-429-6
Online ISBN: 978-1-59745-456-8
eBook Packages: Springer Protocols