Abstract
Multiprotein complexes are important machineries that organize a large number of different proteins into functional units. Studying protein-protein interactions in the complexes, rather than individual proteins, is a fundamental step to gaining functional insights into a biological process. Here, we present the sequential affinity purification and coimmunoprecipitation system that was applied to enable the efficient purification of all the proteins that compose the Lpt system complex in Escherichia coli and their identification by western blotting and mass spectrometry (MS).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2(5):a000414. https://doi.org/10.1101/cshperspect.a000414
Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67(4):593–656. https://doi.org/10.1128/MMBR.67.4.593-656.2003
Luirink J, Yu Z, Wagner S (1817) de Gier JW (2012) Biogenesis of inner membrane proteins in Escherichia coli. Biochim Biophys Acta 6:965–976. https://doi.org/10.1016/j.bbabio.2011.12.006
Imada K (2018) Bacterial flagellar axial structure and its construction. Biophys Rev 10(2):559–570. https://doi.org/10.1007/s12551-017-0378-z
Green ER, Mecsas J (2016) Bacterial secretion systems: an overview. Microbiol Spectr 4(1). https://doi.org/10.1128/microbiolspec.VMBF-0012-2015
Du D, Wang Z, James NR, Voss JE, Klimont E, Ohene-Agyei T, Venter H, Chiu W, Luisi BF (2014) Structure of the AcrAB-TolC multidrug efflux pump. Nature 22 509(7501):512–515. https://doi.org/10.1038/nature13205. Epub 2014 Apr 20. PMID: 24747401; PMCID: PMC4361902
Typas A, Banzhaf M, van den Berg van Saparoea B, Verheul J, Biboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Rechenberg M, Breukink E, den Blaauwen T, Gross CA, Vollmer W (2010) Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 23 143(7):1097–1109. https://doi.org/10.1016/j.cell.2010.11.038
Sperandeo P, Martorana AM, Polissi A (2019) The Lpt ABC transporter for lipopolysaccharide export to the cell surface. Res Microbiol 170(8):366–373. https://doi.org/10.1016/j.resmic.2019.07.005
Bos MP, Robert V, Tommassen J (2007) Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain. EMBO Rep 8(12):1149–1154. https://doi.org/10.1038/sj.embor.7401092
Chng SS, Gronenberg LS, Kahne D (2010) Proteins required for lipopolysaccharide assembly in Escherichia coli form a transenvelope complex. Biochemistry 49(22):4565–4567. https://doi.org/10.1021/bi100493e
Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254
Villa R, Martorana AM, Okuda S, Gourlay LJ, Nardini M, Sperandeo P, Dehò G, Bolognesi M, Kahne D, Polissi A (2013) The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains. J Bacteriol 195(5):1100–1108. https://doi.org/10.1128/JB.02057
Falchi FA, Maccagni EA, Puccio S, Peano C, De Castro C, Palmigiano A, Garozzo D, Martorana AM, Polissi A, Dehò G, Sperandeo P (2017) Mutation and suppressor analysis of the essential lipopolysaccharide transport protein LptA reveals strategies to overcome severe outer membrane permeability defects in Escherichia coli. J Bacteriol 20 200(2):e00487-17. https://doi.org/10.1128/JB.00487-17
Martorana AM, Benedet M, Maccagni EA, Sperandeo P, Villa R, Dehò G, Polissi A (2016) Functional interaction between the cytoplasmic ABC protein LptB and the inner membrane LptC protein, components of the lipopolysaccharide transport machinery in Escherichia coli. J Bacteriol 28 198(16):2192–2203. https://doi.org/10.1128/JB.00329-16
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567. https://doi.org/10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2
Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 1 10(4):1794–1805. https://doi.org/10.1021/pr101065j
Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5(11):976–989. https://doi.org/10.1016/1044-0305(94)80016-2
Babu M, Butland G, Pogoutse O, Li J, Greenblatt JF, Emili A (2009) Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli. Methods Mol Biol 564:373–400. https://doi.org/10.1007/978-1-60761-157-8_22
Wu T, McCandlish AC, Gronenberg LS, Chng SS, Silhavy TJ, Kahne D (2006) Identification of a protein complex that assembles lipopolysaccharide in the outer membrane of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 103:11754–11759
Zeghouf M, Li J, Butland G, Borkowska A, Canadien V, Richards D, Beattie B, Emili A, Greenblatt JF (2004) Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes. J Proteome Res 3(3):463–468. https://doi.org/10.1021/pr034084x
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Martorana, A.M., Santambrogio, C., Polissi, A. (2022). Affinity Purification and Coimmunoprecipitation of Transenvelope Protein Complexes in Gram-Negative Bacteria. In: Sperandeo, P. (eds) Lipopolysaccharide Transport. Methods in Molecular Biology, vol 2548. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2581-1_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2581-1_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2580-4
Online ISBN: 978-1-0716-2581-1
eBook Packages: Springer Protocols