Abstract
Glycans linked to asparagine (N) residues of eukaryotic glycoproteins are typically heterogeneous. This diversity complicates the study of biological functions associated with particular glycan structures and impairs the application of glycoproteins in medicine. Several approaches have been developed to produce homogeneous glycoproteins. We describe a method to produce glycoproteins carrying N-linked N-acetylglucosamine (GlcNAc) through glyco-engineered E. coli cells and enzymatic treatment. N-linked GlcNAc can then be extended by existing methods to produce homogeneous glycoproteins.
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
Similar content being viewed by others
References
Schwarz F, Aebi M (2011) Mechanisms and principles of N-linked protein glycosylation. Curr Opin Struct Biol 21:576–582
Varki A, Cummings RD, Esko JD et al (2009) Essentials of glycobiology. Cold Spring Harbor Press, New York
Rich JR, Withers SG (2009) Emerging methods for the production of homogeneous human glycoproteins. Nat Chem Biol 5:206–215
Castilho A, Gattinger P, Grass J et al (2011) N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans. Glycobiology 21:813–823
Hamilton SR, Bobrowicz P, Bobrowicz B et al (2003) Production of complex human glycoproteins in yeast. Science 301:1244–1246
Hamilton SR, Davidson RC, Sethuraman N et al (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313:1441–1443
Meuris L, Santens F, Elson G et al (2014) GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat Biotechnol 32:485–489
Nagels B, Van Damme EJ, Callewaert N et al (2012) Introduction of tri-antennary N-glycans in Arabidopsis thaliana plants. Plant Sci 185–186:161–168
Schwarz F, Huang W, Li C et al (2010) A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat Chem Biol 6:264–266
Valderrama-Rincon JD, Fisher AC, Merritt JH et al (2012) An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat Chem Biol 8:434–436
Wacker M, Linton D, Hitchen PG et al (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298:1790–1793
Wang LX (2008) Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation. Carbohydr Res 343:1509–1522
Wang LX, Lomino JV (2012) Emerging technologies for making glycan-defined glycoproteins. ACS Chem Biol 7:110–122
Nita-Lazar M, Wacker M, Schegg B et al (2005) The N-X-S/T consensus sequence is required but not sufficient for bacterial N-linked protein glycosylation. Glycobiology 15:361–367
Feldman MF, Wacker M, Hernandez M et al (2005) Engineering N-linked protein glycosylation with diverse O-antigene lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci U S A 102:3016–3021
Kowarik M, Young NM, Numao S et al (2006) Definition of the bacterial N-glycosylation site consensus sequence. EMBO J 25:1957–1966
Fisher AC, Haitjema CH, Guarino C et al (2011) Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Appl Environ Microbiol 77:871–881
Lizak C, Fan YY, Weber TC et al (2011) N-Linked glycosylation of antibody fragments in Escherichia coli. Bioconjug Chem 22:488–496
Mansell TJ, Guarino C, Delisa MP (2013) Engineered genetic selection links in vivo protein folding and stability with asparagine-linked glycosylation. Biotechnol J 8:1445–1451
Perez JM, McGarry MA, Marolda CL et al (2008) Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase. Mol Microbiol 70:1424–1440
Acknowledgments
We thank Yuko Naito-Matsui and Andrea Verhagen for comments on the manuscript. F.S. is the recipient of a postdoctoral fellowship from the Novartis Foundation for medical-biological research. Research in the Aebi Lab is supported by the Swiss National Science Foundation and the ETH Zurich.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Schwarz, F., Aebi, M. (2015). Production of Glycoproteins with Asparagine-Linked N-Acetylglucosamine in Escherichia coli . 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_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2760-9_4
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2759-3
Online ISBN: 978-1-4939-2760-9
eBook Packages: Springer Protocols