Advertisement

A Vector Suite for the Overexpression and Purification of Tagged Outer Membrane, Periplasmic, and Secreted Proteins in E. coli

  • Michael A. Casasanta
  • Daniel J. SladeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1960)

Abstract

Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to challenges with expression and purification. Here we present a suite of vectors for the inducible expression of N-terminally 6His-tagged outer membrane, periplasmic, and secreted proteins in E. coli and show this system to be capable of producing milligram quantities of pure protein for downstream functional and structural analysis. This system can not only be used to purify recombinant virulence factors for structural and functional studies but can also be used to create gain-of-function E. coli for use in phenotypic screens, and examples of each are provided herein.

Keywords

OmpA TamA SufI Periplasm Protein secretion Sec apparatus Twin-arginine translocation Membrane protein Protein purification Protein expression 

Notes

Acknowledgments

Research was supported by start-up funding from the Department of Biochemistry at Virginia Tech, the Institute for Critical Technology and Applied Science (ICTAS) at Virginia Tech, and the National Institute for Food and Agriculture.

References

  1. 1.
    Lee VT, Schneewind O (2001) Protein secretion and the pathogenesis of bacterial infections. Genes Dev 15:1725–1752CrossRefGoogle Scholar
  2. 2.
    Green ER, Mecsas J (2016) Bacterial secretion systems: an overview. In: Kudva IT et al (eds) Virulence mechanisms of bacterial pathogens, 5th edn. American Society of Microbiology, pp 215–239Google Scholar
  3. 3.
    Dautin N, Bernstein HD (2007) Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu Rev Microbiol 61:89–112CrossRefGoogle Scholar
  4. 4.
    Natale P, Brüser T, Driessen AJM (2008) Sec- and tat-mediated protein secretion across the bacterial cytoplasmic membrane--distinct translocases and mechanisms. Biochim Biophys Acta 1778:1735–1756CrossRefGoogle Scholar
  5. 5.
    van den Berg B (2010) Crystal structure of a full-length autotransporter. J Mol Biol 396:627–633CrossRefGoogle Scholar
  6. 6.
    Auclair SM, Bhanu MK, Kendall DA (2012) Signal peptidase I: cleaving the way to mature proteins. Protein Sci 21:13–25CrossRefGoogle Scholar
  7. 7.
    Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234CrossRefGoogle Scholar
  8. 8.
    Casasanta MA, Yoo CC, Smith HB et al (2017) A chemical and biological toolbox for type Vd secretion: characterization of the phospholipase A1 autotransporter FplA from fusobacterium nucleatum. J Biol Chem.  https://doi.org/10.1074/jbc.M117.819144CrossRefGoogle Scholar
  9. 9.
    Woodall CA (2003) Electroporation of E coli. Methods Mol Biol 235:55–69PubMedGoogle Scholar
  10. 10.
    Meuskens I, Michalik M, Chauhan N et al (2017) A new strain collection for improved expression of outer membrane proteins. Front Cell Infect Microbiol 7(464)Google Scholar
  11. 11.
    Smith SM (2011) Strategies for the purification of membrane proteins. Methods Mol Biol 681:485–496CrossRefGoogle Scholar
  12. 12.
    Hobb RI, Fields JA, Burns CM et al (2009) Evaluation of procedures for outer membrane isolation from campylobacter jejuni. Microbiology 155:979–988CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry, Virginia Polytechnic InstituteState UniversityBlacksburgUSA

Personalised recommendations