Skip to main content

A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24 h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.

Key words

  • High-throughput
  • HTP
  • Membrane protein
  • Fluorescent probe
  • Hexahistidine
  • FSEC
  • Detergent

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-9624-7_18
  • Chapter length: 14 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   209.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-9624-7
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   269.00
Price excludes VAT (USA)
Hardcover Book
USD   379.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Yildirim MA, Goh KI, Cusick ME, Barabasi AL, Vidal M (2007) Drug-target network. Nat Biotechnol 25(10):1119–1126. https://doi.org/10.1038/nbt1338

    CAS  CrossRef  PubMed  Google Scholar 

  2. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28(1):235–242

    CAS  CrossRef  Google Scholar 

  3. Gupta K, Donlan JAC, Hopper JTS, Uzdavinys P, Landreh M, Struwe WB, Drew D, Baldwin AJ, Stansfeld PJ, Robinson CV (2017) The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541(7637):421–424. https://doi.org/10.1038/nature20820

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Nicolson GL (2014) The Fluid-Mosaic Model of Membrane Structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years. Biochim Biophys Acta 1838(6):1451–1466. https://doi.org/10.1016/j.bbamem.2013.10.019

    CAS  CrossRef  PubMed  Google Scholar 

  5. Palsdottir H, Hunte C (2004) Lipids in membrane protein structures. Biochim Biophys Acta 1666(1–2):2–18. https://doi.org/10.1016/j.bbamem.2004.06.012

    CAS  CrossRef  PubMed  Google Scholar 

  6. Sonoda Y, Newstead S, Hu NJ, Alguel Y, Nji E, Beis K, Yashiro S, Lee C, Leung J, Cameron AD, Byrne B, Iwata S, Drew D (2011) Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19(1):17–25. https://doi.org/10.1016/j.str.2010.12.001

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Wiener MC (2004) A pedestrian guide to membrane protein crystallization. Methods 34(3):364–372. https://doi.org/10.1016/j.ymeth.2004.03.025

    CAS  CrossRef  PubMed  Google Scholar 

  8. Keyes MH, Gray DN, Kreh KE, Sanders CR (2003) Solubilizing detergents for membrane proteins. In: Iwata S (ed) Methods and results in crystallization of membrane proteins. Sigma-Aldrich, St. Louis, MO, pp 17–38

    Google Scholar 

  9. Ahuja S, Mukund S, Deng L, Khakh K, Chang E, Ho H, Shriver S, Young C, Lin S, Johnson JP Jr, Wu P, Li J, Coons M, Tam C, Brillantes B, Sampang H, Mortara K, Bowman KK, Clark KR, Estevez A, Xie Z, Verschoof H, Grimwood M, Dehnhardt C, Andrez JC, Focken T, Sutherlin DP, Safina BS, Starovasnik MA, Ortwine DF, Franke Y, Cohen CJ, Hackos DH, Koth CM, Payandeh J (2015) Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist. Science 350(6267):aac5464. https://doi.org/10.1126/science.aac5464

    CAS  CrossRef  PubMed  Google Scholar 

  10. Gao Y, Cao E, Julius D, Cheng Y (2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534(7607):347–351. https://doi.org/10.1038/nature17964

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Dorr JM, Scheidelaar S, Koorengevel MC, Dominguez JJ, Schafer M, van Walree CA, Killian JA (2016) The styrene-maleic acid copolymer: a versatile tool in membrane research. Eur Biophys J 45(1):3–21. https://doi.org/10.1007/s00249-015-1093-y

    CAS  CrossRef  PubMed  Google Scholar 

  12. Lee SC, Knowles TJ, Postis VL, Jamshad M, Parslow RA, Lin YP, Goldman A, Sridhar P, Overduin M, Muench SP, Dafforn TR (2016) A method for detergent-free isolation of membrane proteins in their local lipid environment. Nat Protoc 11(7):1149–1162. https://doi.org/10.1038/nprot.2016.070

    CAS  CrossRef  PubMed  Google Scholar 

  13. Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci U S A 93(26):15047–15050

    CAS  CrossRef  Google Scholar 

  14. Denisov IG, Sligar SG (2016) Nanodiscs for structural and functional studies of membrane proteins. Nat Struct Mol Biol 23(6):481–486. https://doi.org/10.1038/nsmb.3195

    CAS  CrossRef  PubMed  Google Scholar 

  15. Drew DE, von Heijne G, Nordlund P, de Gier JW (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507(2):220–224

    CAS  CrossRef  Google Scholar 

  16. Drew D, Sjostrand D, Nilsson J, Urbig T, Chin CN, de Gier JW, von Heijne G (2002) Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis. Proc Natl Acad Sci U S A 99(5):2690–2695. https://doi.org/10.1073/pnas.052018199

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  17. Newstead S, Kim H, von Heijne G, Iwata S, Drew D (2007) High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 104(35):13936–13941. https://doi.org/10.1073/pnas.0704546104

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  18. Hu NJ, Rada H, Rahman N, Nettleship JE, Bird L, Iwata S, Drew D, Cameron AD, Owens RJ (2015) GFP-based expression screening of membrane proteins in insect cells using the baculovirus system. Methods Mol Biol 1261:197–209. https://doi.org/10.1007/978-1-4939-2230-7_11

    CAS  CrossRef  PubMed  Google Scholar 

  19. Chaudhary S, Pak JE, Gruswitz F, Sharma V, Stroud RM (2012) Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat Protoc 7(3):453–466. https://doi.org/10.1038/nprot.2011.453

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  20. Goehring A, Lee CH, Wang KH, Michel JC, Claxton DP, Baconguis I, Althoff T, Fischer S, Garcia KC, Gouaux E (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9(11):2574–2585. https://doi.org/10.1038/nprot.2014.173

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  21. Hays FA, Roe-Zurz Z, Li M, Kelly L, Gruswitz F, Sali A, Stroud RM (2009) Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination. J Struct Funct Genom 10(1):9–16. https://doi.org/10.1007/s10969-008-9046-7

    CAS  CrossRef  Google Scholar 

  22. Drew D, Lerch M, Kunji E, Slotboom DJ, de Gier JW (2006) Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3(4):303–313. https://doi.org/10.1038/nmeth0406-303

    CAS  CrossRef  PubMed  Google Scholar 

  23. Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14(4):673–681. https://doi.org/10.1016/j.str.2006.01.013

    CAS  CrossRef  PubMed  Google Scholar 

  24. Hattori M, Hibbs RE, Gouaux E (2012) A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20(8):1293–1299. https://doi.org/10.1016/j.str.2012.06.009

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  25. Alexander CG, Wanner R, Johnson CM, Breitsprecher D, Winter G, Duhr S, Baaske P, Ferguson N (2014) Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings. Biochim Biophys Acta 1844(12):2241–2250. https://doi.org/10.1016/j.bbapap.2014.09.016

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  26. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3(5):784–798. https://doi.org/10.1038/nprot.2008.44

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  27. Hsieh JM, Besserer GM, Madej MG, Bui HQ, Kwon S, Abramson J (2010) Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini. Protein Sci 19(4):868–880. https://doi.org/10.1002/pro.365

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  28. Backmark AE, Olivier N, Snijder A, Gordon E, Dekker N, Ferguson AD (2013) Fluorescent probe for high-throughput screening of membrane protein expression. Protein Sci 22(8):1124–1132. https://doi.org/10.1002/pro.2297

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  29. Dobrovetsky E, Lu ML, Andorn-Broza R, Khutoreskaya G, Bray JE, Savchenko A, Arrowsmith CH, Edwards AM, Koth CM (2005) High-throughput production of prokaryotic membrane proteins. J Struct Funct Genom 6(1):33–50. https://doi.org/10.1007/s10969-005-1363-5

    CAS  CrossRef  Google Scholar 

  30. Duquesne K, Sturgis JN (2010) Membrane protein solubilization. Methods Mol Biol 601:205–217. https://doi.org/10.1007/978-1-60761-344-2_13

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Jian Payandeh or Christopher M. Koth .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

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

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Xu, H. et al. (2019). A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_18

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

  • eBook Packages: Springer Protocols