Abstract
The bacterial expression of glycoproteins has experienced significant progress in recent years, particularly in regard to the production of conjugate vaccines against pathogens. In this case, a protein carrier conjugated with glycosides is used to produce intense stimulation of the immune system. Glycoconjugate vaccines account for 35% of the global vaccine market, and consequently, several biotechnological companies have developed products for the purification of glycosylated proteins to attain homogeneity. In this chapter we present a general process for glycoprotein production in Escherichia coli and a practice method for purification of glycosylated proteins, using affinity chromatography.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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
Baker JL, Çelik E, DeLisa MP (2013) Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation. Trends Biotechnol 31:313–323
Li H, Debowski AW, Liao T et al (2017) Understanding protein glycosylation pathways in bacteria. Future Microbiol 12:59–72
Feldman MF, Wacker M, Hernandez M et al (2005) Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci 102:3016–3021
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
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
Ravenscroft N, Haeuptle MA, Kowarik M et al (2016) Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli. Glycobiology 26:51–62
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
Ihssen J, Kowarik M, Dilettoso S et al (2010) Production of glycoprotein vaccines in Escherichia coli. Microb Cell Fact 9:61
Garcia-Quintanilla F, Iwashkiw JA, Price NL et al (2014) Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery. Front Microbiol 5:381
Wacker M, Wang L, Kowarik M et al (2014) Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli. J Infect Dis 209:1551–1561
Lam SK, Ng TB (2011) Lectins: production and practical applications. Appl Microbiol Biotechnol 89:45–55
Friedman AM, Long SR, Brown SE et al (1982) Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296
Simonian MH (2002) Spectrophotometric determination of protein concentration. Curr Protoc Cell Biol Appendix 3:Appendix 3B
Kruger NJ (2002) The Bradford method for protein quantitation. In: Protein protoc handbook. Humana Press, New Jersey, pp 15–22
Pohleven J, Trukelj B, Kos J (2012) Affinity chromatography of lectins. In: Affinity chromatography. InTech, Rijeka, Croatia, pp 49–74
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Breyer, C.A., de Oliveira, M.A., Pessoa, A. (2018). Expression of Glycosylated Proteins in Bacterial System and Purification by Affinity Chromatography. In: Picanço-Castro, V., Swiech, K. (eds) Recombinant Glycoprotein Production. Methods in Molecular Biology, vol 1674. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7312-5_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7312-5_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7311-8
Online ISBN: 978-1-4939-7312-5
eBook Packages: Springer Protocols