Abstract
Integral outer membrane proteins (OMPs) play key roles in solute transport, adhesion, and other processes. In Neisseria, they can also function as major protective antigens. Structural, biophysical, and immunological studies of Neisserial OMPs require their isolation in milligram quantities. Purification of any OMP directly from Neisseria would require the growth of large quantities of cell mass, with attendant concerns about safety and convenience. As a result, many investigators have developed methods for expression of OMPs into inclusion bodies in E. coli, followed by refolding of the resolubilized protein. Here we describe such a method, as optimized for the PorA porin but which can be applied, with suitable adaptation, to other OMPs. We also describe an approach to the crystallization of PorA.
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References
Wimley WC (2003) The versatile beta-barrel membrane protein. Curr Opin Struct Biol 13: 404–411.
Jansen C, Wiese A, Reubsaet L et al (2000) Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis. Biochim Biophys Acta-Biomem 1464: 284–298.
Tanabe M, Nimigean CM, Iverson TM (2010) Structural basis for solute transport, nucleotide regulation, and immunological recognition of Neisseria meningitidis PorB. Proc Natl Acad Sci USA 107: 6811–6816.
Kortekaas J, Muller SA, Ringler P et al (2006) Immunogenicity and structural characterisation of an in vitro folded meningococcal siderophore receptor (FrpB, FetA). Microb Infect 8: 2145–2153.
Vandeputte-Rutten L, Bos MP, Tommassen J et al (2003) Crystal structure of Neisserial surface protein A (NspA), a conserved outer membrane protein with vaccine potential. J Biol Chem 278: 24825–24830.
Prince SM, Achtman M, Derrick JP (2002) Crystal structure of the OpcA integral membrane adhesin from Neisseria meningitidis Proc Natl Acad Sci USA 99: 3417–3421.
Moore J, Bailey SES, Benmechernene Z et al (2005) Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis J Biol Chem 280: 31489–31497.
Feavers IM, Pizza M (2009) Meningococcal protein antigens and vaccines. Vaccine 27: B42–B50.
Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 346–356.
Newstead S, Ferrandon S, Iwata S (2008) Rationalizing alpha-helical membrane protein crystallization. Prot Sci 17: 466–472.
Gorrec F (2009) The MORPHEUS protein crystallization screen. J Applied Crystallog 42: 1035–1042.
Acknowledgments
We thank Dr Hannah Chan and Prof. Ian Feavers (National Institute for Biological Standards and Control, South Mimms) for supply of PorA expression vectors.
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© 2012 Springer Science+Business Media, LLC
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Saleem, M., Moore, J., Derrick, J.P. (2012). Expression, Purification, and Crystallization of Neisserial Outer Membrane Proteins. In: Christodoulides, M. (eds) Neisseria meningitidis. Methods in Molecular Biology, vol 799. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-346-2_6
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DOI: https://doi.org/10.1007/978-1-61779-346-2_6
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Publisher Name: Humana, Totowa, NJ
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