Expression of Prokaryotic Integral Membrane Proteins in E. coli

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

Production of prokaryotic membrane proteins for structural and functional studies in E. coli can be parallelized and miniaturized. All stages from cloning, expression, purification to detergent selection can be investigated using high-throughput techniques to rapidly and economically find tractable targets.

Key words

High throughput Membrane proteins Detergent assay Multiwell 

References

  1. 1.
    Kloppmann E, Punta M, Rost B (2012) Structural genomics plucks high-hanging membrane proteins. Curr Opin Struct Biol 22:326–332CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Willis MS, Koth CM (2008) Structural proteomics of membrane proteins: a survey of published techniques and design of a rational high throughput strategy. Methods Mol Biol 426:277–295CrossRefPubMedGoogle Scholar
  3. 3.
    Punta M, Love J, Handelman S et al (2009) Structural genomics target selection for the New York consortium on membrane protein structure. J Struct Funct Genomics 10:255–268CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Eshaghi S (2009) High-throughput expression and detergent screening of integral membrane proteins. Methods Mol Biol 498:265–271CrossRefPubMedGoogle Scholar
  5. 5.
    Love J, Mancia F, Shapiro L et al (2010) The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins. J Struct Funct Genomics 11:191–199CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Graslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146CrossRefPubMedGoogle Scholar
  7. 7.
    Xiao R, Anderson S, Aramini J et al (2010) The high-throughput protein sample production platform of the northeast structural genomics consortium. J Struct Biol 172:21–33CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chaudhary S, Pak JE, Pedersen BP et al (2011) Efficient expression screening of human membrane proteins in transiently transfected human embryonic kidney 293S cells. Methods 55:273–280CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Li M, Hays FA, Roe-Zurz Z et al (2009) Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening. J Mol Biol 385:820–830CrossRefPubMedGoogle Scholar
  10. 10.
    Mancia F, Love J (2010) High-throughput expression and purification of membrane proteins. J Struct Biol 172:85–93CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lewinson O, Lee AT, Rees DC (2008) The funnel approach to the pre-crystallization production of membrane proteins. J Mol Biol 377:62–73CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gustafsson C, Minshull J, Govindarajan S et al (2012) Engineering genes for predictable protein expression. Protein Expr Purif 83:37–46CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Savitsky P, Bray J, Cooper CDO et al (2010) High-throughput production of human proteins for crystallization: the SGC experience. J Struct Biol 172:3–13CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Eschenfeldt WH, Lucy S, Millard CS et al (2009) A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol Biol 498:105–115CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Everett JK, Acton TB, Montelione GT (2004) Primer Prim'er: a web based server for automated primer design. J Struct Funct Genomics 5:13–21CrossRefPubMedGoogle Scholar
  16. 16.
    Zielinski D, Gordon A, Zaks BL et al (2014) iPipet: sample handling using a tablet. Nat Methods 11:784–785CrossRefPubMedGoogle Scholar
  17. 17.
    Newby ZE, O’Connell JD, Gruswitz F et al (2009) A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat Protoc 4:619–637CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Bernard P, Couturier M (1992) Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J Mol Biol 226:735–745CrossRefPubMedGoogle Scholar
  19. 19.
    Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234CrossRefPubMedGoogle Scholar
  20. 20.
    Rath A, Glibowicka M, Nadeau VG et al (2009) Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A 106:1760–1765CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Slotboom DJ, Duurkens RH, Olieman K et al (2008) Static light scattering to characterize membrane proteins in detergent solution. Methods 46:73–82CrossRefPubMedGoogle Scholar
  22. 22.
    Miercke LJ, Robbins RA, Stroud RM (2014) Tetra detector analysis of membrane proteins. Curr Protoc Protein Sci 77:1–30Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of BiochemistryAlbert Einstein College of Medicine at Yeshiva UniversityBronxUSA
  2. 2.ATUMNewarkUSA

Personalised recommendations