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Affinity Purification and Coimmunoprecipitation of Transenvelope Protein Complexes in Gram-Negative Bacteria

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Lipopolysaccharide Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2548))

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).

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References

  1. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 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

    Article  CAS  Google Scholar 

  4. 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

    Article  CAS  PubMed  Google Scholar 

  5. Green ER, Mecsas J (2016) Bacterial secretion systems: an overview. Microbiol Spectr 4(1). https://doi.org/10.1128/microbiolspec.VMBF-0012-2015

  6. 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

    Article  CAS  Google Scholar 

  7. 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

    Article  CAS  Google Scholar 

  8. 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

    Article  CAS  PubMed  Google Scholar 

  9. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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

    Article  CAS  PubMed  Google Scholar 

  11. Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254

    Article  CAS  Google Scholar 

  12. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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

    Article  Google Scholar 

  14. 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

    Article  CAS  Google Scholar 

  15. 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

    Article  CAS  PubMed  Google Scholar 

  16. 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

    Article  CAS  Google Scholar 

  17. 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

    Article  CAS  PubMed  Google Scholar 

  18. 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

    Article  CAS  PubMed  Google Scholar 

  19. 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

    Article  CAS  Google Scholar 

  20. 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

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Alessandra M. Martorana .

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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

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  • DOI: https://doi.org/10.1007/978-1-0716-2581-1_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2580-4

  • Online ISBN: 978-1-0716-2581-1

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