Advertisement

Membrane Protein Production in Escherichia coli

  • Benjamin C. McIlwainEmail author
  • Ali A. KermaniEmail author
Protocol
  • 442 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)

Abstract

Escherichia coli is the workhorse of the structural biology lab. In addition to routine cloning and molecular biology, E. coli can be used as a factory for the production of recombinant membrane proteins. Purification of homogeneous samples of membrane protein expressed in E. coli is a significant bottleneck for researchers, and the protocol we present here for the overexpression and purification of membrane proteins in E. coli will provide a solid basis to develop lab- and protein-specific protocols for your membrane protein of interest. We additionally provide extensive notes on the purification process, as well as the theory surrounding principles of purification.

Key words

Membrane protein E. coli Crystallography Ion channel Transporter. 

Notes

Acknowledgment

We would like to thank Randy Stockbridge as well as other members of the Stockbridge lab for helpful suggestions, guidance, and feedback on this chapter.

References

  1. 1.
    Wallin E, Heijne GV (1998) Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 7(4):1029–1038PubMedCrossRefGoogle Scholar
  2. 2.
    Stockbridge RB, Lim HH, Otten R, Williams C, Shane T, Weinberg Z, Miller C (2012) Fluoride resistance and transport by riboswitch-controlled CLC antiporters. Proc Natl Acad Sci 109(38):15289–15294PubMedCrossRefGoogle Scholar
  3. 3.
    Stockbridge RB, Robertson JL, Kolmakova-Partensky L, Miller C (2013) A family of fluoride-specific ion channels with dual-topology architecture. elife 2:e01084PubMedCrossRefGoogle Scholar
  4. 4.
    Kermani AA, Macdonald CB, Gundepudi R, Stockbridge RB (2018) Guanidinium export is the primal function of SMR family transporters. Proc Natl Acad Sci 115(12):3060–3065PubMedCrossRefGoogle Scholar
  5. 5.
    McIlwain BC, Newstead S, Stockbridge RB (2018) Cork-in-bottle occlusion of fluoride ion channels by crystallization chaperones. Structure 26(4):635–639PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Marshall SS, Niesen MJ, Müller A, Tiemann K, Saladi SM, Galimidi RP, Zhang B, Clemons WM Jr, Miller TF III (2016) A link between integral membrane protein expression and simulated integration efficiency. Cell Rep 16(8):2169PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16(13):6127–6145PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Wagner S, Bader ML, Drew D, de Gier JW (2006) Rationalizing membrane protein overexpression. Trends Biotechnol 24(8):364–371PubMedCrossRefGoogle Scholar
  9. 9.
    Hartley JL (2006) Cloning technologies for protein expression and purification. Curr Opin Biotechnol 17(4):359–366PubMedCrossRefGoogle Scholar
  10. 10.
    Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189(1):113–130PubMedCrossRefGoogle Scholar
  11. 11.
    Zhang X, Studier FW (1997) Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol 269(1):10–27PubMedCrossRefGoogle Scholar
  12. 12.
    Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260(3):289–298PubMedCrossRefGoogle Scholar
  13. 13.
    Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M, Högbom M, Van Wijk KJ, Slotboom DJ, Persson JO, De Gier JW (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci 105(38):14371–14376PubMedCrossRefGoogle Scholar
  14. 14.
    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(4):648–659PubMedCrossRefGoogle Scholar
  15. 15.
    Kohanski MA, Dwyer DJ, Collins JJ (2010) How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol 8(6):423PubMedCrossRefGoogle Scholar
  16. 16.
    Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophy Acta 1666(1–2):105–117CrossRefGoogle Scholar
  17. 17.
    Garavito RM, Ferguson-Miller S (2001) Detergents as tools in membrane biochemistry. J Biol Chem 276(35):32403–32406PubMedCrossRefGoogle Scholar
  18. 18.
    Anandan A, Vrielink A (2016) Detergents in membrane protein purification and crystallisation. In: The Next Generation in Membrane Protein Structure Determination. Springer, Cham, pp 13–28CrossRefGoogle Scholar
  19. 19.
    Schimerlik MI (1998) Overview of membrane protein solubilization. Curr Protoc Neurosci 2(1):5–9CrossRefGoogle Scholar
  20. 20.
    Wang H, Elferich J, Gouaux E (2012) Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context. Nat Struct Mol Biol 19(2):212PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Quick M, Shi L, Zehnpfennig B, Weinstein H, Javitch JA (2012) Experimental conditions can obscure the second high-affinity site in LeuT. Nat Struct Mol Biol 19(2):207PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14(4):673–681PubMedCrossRefGoogle Scholar
  23. 23.
    Hattori M, Hibbs RE, Gouaux E (2012) A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20(8):1293–1299PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Bird LE, Rada H, Verma A, Gasper R, Birch J, Jennions M, Lӧwe J, Moraes I, Owens RJ (2015) Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli. J Vis Exp 95:e52357Google Scholar
  25. 25.
    Kosobokova EN, Skrypnik KA, Kosorukov VS (2016) Overview of fusion tags for recombinant proteins. Biochem Mosc 81(3):187–200CrossRefGoogle Scholar
  26. 26.
    Waugh DS (2011) An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif 80(2):283–293PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Molecular, Cellular, and Developmental BiologyUniversity of MichiganAnn ArborUSA

Personalised recommendations