SpringerLink
Log in

Bacterial Envelope Fractionation

  • Protocol
  • First Online:
Transmembrane β-Barrel Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2778))

Abstract

Numerous bioinformatics tools allow predicting the localization of membrane proteins in the outer or inner membrane of Escherichia coli with high precision. Nevertheless, it might be desirable to experimentally verify such predictions or to assay the correct localization of recombinant or mutated variants of membrane proteins. Here we describe two methods (preferential detergent solubilization and sucrose-gradient fractionation) that allow to fractionate Gram-negative bacterial membranes and subsequently to enrich inner or outer membrane proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Miller SI, Salama NR (2018) The gram-negative bacterial periplasm: size matters. PLoS Biol 16:e2004935. https://doi.org/10.1371/journal.pbio.2004935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2:a000414. https://doi.org/10.1101/cshperspect.a000414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. DiRienzo JM, Nakamura K, Inouye M (1978) The outer membrane proteins of Gram-negative bacteria: biosynthesis, assembly, and functions. Annu Rev Biochem 47:481–532. https://doi.org/10.1146/annurev.bi.47.070178.002405

    Article  CAS  PubMed  Google Scholar 

  4. Osborn MJ, Gander JE, Parisi E, Carson J (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247:3962–3972

    Article  CAS  PubMed  Google Scholar 

  5. Strauss J, Burnham NA, Camesano TA (2009) Atomic force microscopy study of the role of LPS O-antigen on adhesion of E. coli. J Mol Recognit 22:347–355. https://doi.org/10.1002/jmr.955

    Article  CAS  PubMed  Google Scholar 

  6. Zgurskaya HI, Lopez CA, Gnanakaran S (2015) Permeability barrier of gram-negative cell envelopes and approaches to bypass it. ACS Infect Dis 1:512–522. https://doi.org/10.1021/acsinfecdis.5b00097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Delcour AH (2009) Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta 1794:808–816. https://doi.org/10.1016/j.bbapap.2008.11.005

    Article  CAS  PubMed  Google Scholar 

  8. Braun V (1975) Covalent lipoprotein from the outer membrane of Escherichia coli. Biochim Biophys Acta 415:335–377. https://doi.org/10.1016/0304-4157(75)90013-1

    Article  CAS  PubMed  Google Scholar 

  9. Almen MS, Nordstrom KJ, Fredriksson R, Schioth HB (2009) Map** the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC Biol 7:50. https://doi.org/10.1186/1741-7007-7-50

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wallin E, von Heijne G (1998) Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 7:1029–1038. https://doi.org/10.1002/pro.5560070420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580. https://doi.org/10.1006/jmbi.2000.4315

    Article  CAS  PubMed  Google Scholar 

  12. Sueki A, Stein F, Savitski MM, Selkrig J, Typas A (2020) Systematic localization of Escherichia coli membrane proteins. mSystems 5. https://doi.org/10.1128/mSystems.00808-19

  13. Wimley WC (2003) The versatile beta-barrel membrane protein. Curr Opin Struct Biol 13:404–411. https://doi.org/10.1016/s0959-440x(03)00099-x

    Article  CAS  PubMed  Google Scholar 

  14. Wimley WC (2002) Toward genomic identification of beta-barrel membrane proteins: composition and architecture of known structures. Protein Sci 11:301–312. https://doi.org/10.1110/ps.29402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. von Heijne G (1999) Recent advances in the understanding of membrane protein assembly and structure. Q Rev Biophys 32:285–307. https://doi.org/10.1017/s0033583500003541

    Article  Google Scholar 

  16. Rosas NC, Lithgow T (2022) Targeting bacterial outer-membrane remodelling to impact antimicrobial drug resistance. Trends Microbiol 30:544–552. https://doi.org/10.1016/j.tim.2021.11.002

    Article  CAS  PubMed  Google Scholar 

  17. WHO (2021) Antimicrobial resistance. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

  18. Lang H (2000) Outer membrane proteins as surface display systems. Int J Med Microbiol 290:579–585. https://doi.org/10.1016/S1438-4221(00)80004-1

    Article  CAS  PubMed  Google Scholar 

  19. Massari P, Ram S, Macleod H, Wetzler LM (2003) The role of porins in neisserial pathogenesis and immunity. Trends Microbiol 11:87–93. https://doi.org/10.1016/s0966-842x(02)00037-9

    Article  CAS  PubMed  Google Scholar 

  20. Liu C, Chen Z, Tan C, Liu W, Xu Z, Zhou R, Chen H (2012) Immunogenic characterization of outer membrane porins OmpC and OmpF of porcine extraintestinal pathogenic Escherichia coli. FEMS Microbiol Lett 337:104–111. https://doi.org/10.1111/1574-6968.12013

    Article  CAS  PubMed  Google Scholar 

  21. Schlegel S, Hjelm A, Baumgarten T, Vikstrom D, de Gier JW (2014) Bacterial-based membrane protein production. Biochim Biophys Acta 1843:1739–1749. https://doi.org/10.1016/j.bbamcr.2013.10.023

    Article  CAS  PubMed  Google Scholar 

  22. Meuskens I, Michalik M, Chauhan N, Linke D, Leo JC (2017) A new strain collection for improved expression of outer membrane proteins. Front Cell Infect Microbiol 7:464. https://doi.org/10.3389/fcimb.2017.00464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Orfanoudaki G, Economou A (2014) Proteome-wide subcellular topologies of E. coli polypeptides database (STEPdb). Mol Cell Proteomics 13:3674–3687. https://doi.org/10.1074/mcp.O114.041137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Thein M, Sauer G, Paramasivam N, Grin I, Linke D (2010) Efficient subfractionation of gram-negative bacteria for proteomics studies. J Proteome Res 9:6135–6147. https://doi.org/10.1021/pr1002438

    Article  CAS  PubMed  Google Scholar 

  25. Miura T, Mizushima S (1968) Separation by density gradient centrifugation of two types of membranes from spheroplast membrane of Escherichia coli K12. Biochim Biophys Acta 150:159–161. https://doi.org/10.1016/0005-2736(68)90020-5

    Article  CAS  PubMed  Google Scholar 

  26. Schnaitman CA (1971) Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol 108:545–552. https://doi.org/10.1128/jb.108.1.545-552.1971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Filip C, Fletcher G, Wulff JL, Earhart CF (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115:717–722. https://doi.org/10.1128/jb.115.3.717-722.1973

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Edwards GB, Muthurajan UM, Bowerman S, Luger K (2020) Analytical ultracentrifugation (AUC): an overview of the application of fluorescence and absorbance AUC to the study of biological macromolecules. Curr Protoc Mol Biol 133:e131. https://doi.org/10.1002/cpmb.131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Laskowska E, Bohdanowicz J, Kuczynska-Wisnik D, Matuszewska E, Kedzierska S, Taylor A (2004) Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells. Microbiology (Reading) 150:247–259. https://doi.org/10.1099/mic.0.26470-0

    Article  CAS  PubMed  Google Scholar 

  30. Arachea BT, Sun Z, Potente N, Malik R, Isailovic D, Viola RE (2012) Detergent selection for enhanced extraction of membrane proteins. Protein Expr Purif 86:12–20. https://doi.org/10.1016/j.pep.2012.08.016

    Article  CAS  PubMed  Google Scholar 

  31. Mathieu K, Javed W, Vallet S, Lesterlin C, Candusso MP, Ding F, Xu XN, Ebel C, Jault JM, Orelle C (2019) Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain. Sci Rep 9:2654. https://doi.org/10.1038/s41598-019-39382-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. https://doi.org/10.1016/0003-2697(85)90442-7

    Article  CAS  PubMed  Google Scholar 

  33. Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22. https://doi.org/10.1038/nprot.2006.4

    Article  CAS  PubMed  Google Scholar 

  34. Islam MS, Aryasomayajula A, Selvaganapathy PR (2017) A review on macroscale and microscale cell lysis methods. Micromachines 8:83. https://doi.org/10.3390/mi8030083

    Article  Google Scholar 

  35. Beis K, Whitfield C, Booth I, Naismith JH (2006) Two-step purification of outer membrane proteins. Int J Biol Macromol 39:10–14. https://doi.org/10.1016/j.ijbiomac.2005.12.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rampado R, Giordano F, Moracci L, Crotti S, Caliceti P, Agostini M, Taraballi F (2022) Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells. J Pharm Biomed Anal 219:114926. https://doi.org/10.1016/j.jpba.2022.114926

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Research Council of Norway, Grant 294605 (Center for Digital Life).

Author information

Authors and Affiliations

  1. Department of Biosciences, University of Oslo, Oslo, Norway

    Athanasios Saragliadis & Dirk Linke

Authors
  1. Athanasios Saragliadis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dirk Linke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Athanasios Saragliadis .

Editor information

Editors and Affiliations

  1. Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative, CNRS, Université de Toulouse, Toulouse, France

    Raffaele Ieva

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Saragliadis, A., Linke, D. (2024). Bacterial Envelope Fractionation. In: Ieva, R. (eds) Transmembrane β-Barrel Proteins. Methods in Molecular Biology, vol 2778. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3734-0_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3734-0_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3733-3

  • Online ISBN: 978-1-0716-3734-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation