Large Complexes: Cloning Strategy, Production, and Purification

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

Membrane proteins can assemble and form complexes in the cell envelope. In Gram-negative bacteria, a number of multiprotein complexes, including secretion systems, efflux pumps, molecular motors, and pilus assembly machines, comprise proteins from the inner and outer membranes. Besides the structures of isolated soluble domains, only a few atomic structures of these assembled molecular machines have been elucidated. To better understand the function and to solve the structure of protein complexes, it is thus necessary to design dedicated production and purification processes. Here we present cloning procedures to overproduce membrane proteins into Escherichia coli cells and describe the cloning and purification strategy for the Type VI secretion TssJLM membrane complex.

Key words

Membrane protein complexes Escherichia coli T7 overexpression Protein purification 

Notes

Acknowledgements

We would like to thank M. Petiti, L. Houot, H. Célia, and D. Duché for their careful reading of the text. E.D. and R.L. are funded by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and two grants from the Agence Nationale de la Recherche (ANR-10-JCJC-1303-03 and ANR-14-CE09-0023, respectively). ED is supported by the Institut National de la Santé Et de la Recherche Médicale through a permanent research position.

References

  1. 1.
    Amann E, Brosius J, Ptashne M (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25:167–178CrossRefPubMedGoogle Scholar
  2. 2.
    Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res 25:1203–1210CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Cagnon C, Valverde V, Masson JM (1991) A new family of sugar-inducible expression vectors for Escherichia coli. Protein Eng 4:843–847CrossRefPubMedGoogle Scholar
  4. 4.
    Skerra A (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151:131–135CrossRefPubMedGoogle Scholar
  5. 5.
    Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Haldimann A, Daniels LL, Wanner BL (1998) Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J Bacteriol 180:1277–1286PubMedPubMedCentralGoogle Scholar
  7. 7.
    Balzer S, Kucharova V, Megerle J, Lale R, Brautaset T, Valla S (2013) A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli. Microb Cell Fact 12:26CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219:37–44CrossRefPubMedGoogle Scholar
  9. 9.
    Schlegel S, Löfblom J, Lee C, Hjelm A, Klepsch M, Strous M, Drew D, Slotboom DJ, de Gier JW (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J Mol Biol 423:648–659CrossRefPubMedGoogle Scholar
  10. 10.
    Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45–59CrossRefPubMedGoogle Scholar
  11. 11.
    Schwarz D, Junge F, Durst F, Frölich N, Schneider B, Reckel S, Sobhanifar S, Dötsch V, Bernhard F (2007) Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc 2:2945–2957CrossRefPubMedGoogle Scholar
  12. 12.
    Guihard G, Boulanger P, Bénédetti H, Lloubes R, Besnard M, Letellier L (1994) Colicin A and the Tol proteins involved in its translocation are preferentially located in the contact sites between the inner and outer membranes of Escherichia coli cells. J Biol Chem 269:5874–5880PubMedGoogle Scholar
  13. 13.
    Cascales E, Lloubes R, Sturgis JN (2001) The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB. Mol Microbiol 42:795–807CrossRefPubMedGoogle Scholar
  14. 14.
    Celia H, Noinaj N, Zakharov SD, Bordignon E, Botos I, Santamaria M, Barnard TJ, Cramer WA, Lloubes R, Buchanan SK (2016) Structural insight into the role of the Ton complex in energy transduction. Nature 538:60–65CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Pramanik A, Zhang F, Schwarz H, Schreiber F, Braun V (2010) ExbB protein in the cytoplasmic membrane of Escherichia coli forms a stable oligomer. Biochemistry 49:8721–8728CrossRefPubMedGoogle Scholar
  16. 16.
    Pramanik A, Hauf W, Hoffmann J, Cernescu M, Brutschy B, Braun V (2011) Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry. Biochemistry 50:8950–8956CrossRefPubMedGoogle Scholar
  17. 17.
    Sverzhinsky A, Fabre L, Cottreau AL, Biot-Pelletier DM, Khalil S, Bostina M, Rouiller I, Coulton JW (2014) Coordinated rearrangements between cytoplasmic and periplasmic domains of the membrane protein complex ExbB-ExbD of Escherichia coli. Structure 22:791–797CrossRefPubMedGoogle Scholar
  18. 18.
    Yonekura K, Maki-Yonekura S, Homma M (2011) Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation. J Bacteriol 193:3863–3870CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kim JS, Jeong H, Song S, Kim HY, Lee K, Hyun J, Ha NC (2015) Structure of the tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode. Mol Cells 38:180–186CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, Lu F, Redzej A, Fronzes R, Orlova EV, Waksman G (2014) Structure of a type IV secretion system. Nature 508:550–553CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Durand E, Nguyen VS, Zoued A, Logger L, Péhau-Arnaudet G, Aschtgen MS, Spinelli S, Desmyter A, Bardiaux B, Dujeancourt A, Roussel A, Cambillau C, Cascales E, Fronzes R (2015) Biogenesis and structure of a type VI secretion membrane core complex. Nature 523:555–560CrossRefPubMedGoogle Scholar
  22. 22.
    Bakelar J, Buchanan SK, Noinaj N (2016) The structure of the β-barrel assembly machinery complex. Science 351:180–186CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Unger T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172:34–44CrossRefPubMedGoogle Scholar
  24. 24.
    Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A 82:1074–1078CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130CrossRefPubMedGoogle Scholar
  26. 26.
    Narayanan A, Ridilla M, Yernool DA (2011) Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions. Protein Sci 20:51–61CrossRefPubMedGoogle Scholar
  27. 27.
    Stuchlík S, Turna J (1998) Overexpression of the FNR protein of Escherichia coli with T7 expression system. Folia Microbiol (Praha) 43:601–604CrossRefGoogle Scholar
  28. 28.
    Doherty AJ, Connolly BA, Worrall AF (1993) Overproduction of the toxic protein, bovine pancreatic DNaseI, in Escherichia coli using a tightly controlled T7-promoter-based vector. Gene 136:337–340CrossRefPubMedGoogle Scholar
  29. 29.
    van den Ent F, Löwe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74CrossRefPubMedGoogle Scholar
  30. 30.
    Jeong J, Yim H, Ryu J, Lee H, Lee J, Seen D, Kang S (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78:5440–5443CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bénédetti H, Lazdunski C, Lloubes R (1991) Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system. EMBO J 10:1989–1995PubMedPubMedCentralGoogle Scholar
  32. 32.
    Derouiche R, Bénédetti H, Lazzaroni JC, Lazdunski C, Lloubes R (1995) Protein complex within Escherichia coli inner membrane. TolA N-terminal domain interacts with TolQ and TolR proteins. J Biol Chem 270:11078–11084CrossRefPubMedGoogle Scholar
  33. 33.
    Konovalova A, Silhavy TJ (2015) Outer membrane lipoprotein biogenesis: Lol is not the end. Philos Trans R Soc Lond B Biol Sci 370:1679CrossRefGoogle Scholar
  34. 34.
    Ringquist S, Shinedling S, Barrick D, Green L, Binkley J, Stormo GD, Gold L (1992) Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol Microbiol 6:1219–1229CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM, UMR7255)Institut de Microbiologie de la Méditerranée (IMM), Aix-Marseille Univ and CNRSMarseille cedex 20France

Personalised recommendations