High-Yield Preparation of Outer Membrane Protein Efflux Pumps by in Vitro Refolding is Concentration Dependent


Overexpression of tripartite efflux pump systems in gram-negative bacteria is a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields, requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. A classical method for refolding outer membrane proteins involves unfolding inclusion bodies in urea followed by refolding in lipid or detergent micelles. However, that method has not yet been successful in refolding tripartite efflux pump TolC. Here, we find that refolding TolC from inclusion bodies requires an additional oligomerization enhancing step of sample concentration. We show that by our method of refolding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that TolC refolding is concentration dependent. We then extended our method to refolding CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in refolding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.

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  1. Agarwal R, Zakharov S, Hasan SS, Ryan CM, Whitelegge JP, Cramer WA (2014) Structure-function of cyanobacterial outer-membrane protein, Slr1270: homolog of Escherichiacoli drug export/colicin import protein, TolC. FEBS Lett 588(21):3793–3801

    CAS  Article  Google Scholar 

  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402

    CAS  Article  Google Scholar 

  3. Altschul SF, Wootton JC, Gertz EM, Agarwala R, Morgulis A, Schaffer AA, Yu YK (2005) Protein database searches using compositionally adjusted substitution matrices. FEBS J 272(20):5101–5109

    CAS  Article  Google Scholar 

  4. Bannwarth M, Schulz GE (2003) The expression of outer membrane proteins for crystallization. Biochim Biophys Acta 1610(1):37–45

    CAS  Article  Google Scholar 

  5. Buchanan SK (1999) Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. Curr Opin Struct Biol 9(4):455–461

    CAS  Article  Google Scholar 

  6. Burgess NK, Dao TP, Stanley AM, Fleming KG (2008) Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro. J Biol Chem 283(39):26748–26758

  7. Charbonnier F, Kohler T, Pechere JC, Ducruix A (2001) Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of pseudomonas aeruginosa. Protein Expr Purif 23(1):121–127

    CAS  Article  Google Scholar 

  8. Daury L, Orange F, Taveau JC, Verchere A, Monlezun L, Gounou C, Marreddy RK, Picard M, Broutin I, Pos KM, Lambert O (2016) Tripartite assembly of RND multidrug efflux pumps. Nat Commun 7:10731

    CAS  Article  Google Scholar 

  9. Franklin MW, Nepomnyachiy S, Feehan R, Ben-Tal N, Kolodny R, Slusky JSG (2018) Efflux pumps represent possible evolutionary convergence onto the beta-barrel fold. Structure 26(9):1266-1274.e1262

    CAS  Article  Google Scholar 

  10. Hagn F, Nasr ML, Wagner G (2018) Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc 13(1):79–98

    CAS  Article  Google Scholar 

  11. Heller KB (1978) Apparent molecular weights of a heat-modifiable protein from the outer membrane of Escherichia coli in gels with different acrylamide concentrations. J Bacteriol 134(3):1181–1183

  12. Horna G, Lopez M, Guerra H, Saenz Y, Ruiz J (2018) Interplay between MexAB-OprM and MexEF-OprN in clinical isolates of pseudomonas aeruginosa. Sci Rep 8(1):16463

    Article  Google Scholar 

  13. Koronakis V, Li J, Koronakis E, Stauffer K (1997) Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals. Mol Microbiol 23(3):617–626

    CAS  Article  Google Scholar 

  14. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C (2000) Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405(6789):914–919

    CAS  Article  Google Scholar 

  15. Mastronarde DN (2018) Advanced data acquisition from electron microscopes with serialEM. Microsc Microanal 24(S1):864–865

    Article  Google Scholar 

  16. Nakamura K, Mizushima S (1976) Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichiacoli K-12. J Biochem 80(6):1411–1422

    CAS  Article  Google Scholar 

  17. Neuberger A, Du D, Luisi BF (2018) Structure and mechanism of bacterial tripartite efflux pumps. Res Microbiol 169(7–8):401–413

    CAS  Article  Google Scholar 

  18. Noinaj N, Kuszak AJ, Buchanan SK (2015) Heat modifiability of outer membrane proteins from gram-negative bacteria. Methods Mol Biol 1329:51–56

    CAS  Article  Google Scholar 

  19. Noinaj N, Mayclin S, Stanley AM, Jao CC, Buchanan SK (2016) From constructs to crystals - towards structure determination of beta-barrel outer membrane proteins. J Vis Exp. https://doi.org/10.3791/53245

    Article  PubMed  PubMed Central  Google Scholar 

  20. Pilsl H, Braun V (1995) Evidence that the immunity protein inactivates colicin 5 immediately prior to the formation of the transmembrane channel. J Bacteriol 177(23):6966–6972

    CAS  Article  Google Scholar 

  21. Popot JL (2014) Folding membrane proteins in vitro: a table and some comments. Arch Biochem Biophys 564:314–326

    CAS  Article  Google Scholar 

  22. Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14(3):290–296

    CAS  Article  Google Scholar 

  23. Rohou A, Grigorieff N (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs. J Struct Biol 192(2):216–221

    Article  Google Scholar 

  24. Rosenbusch JP (1974) Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J Biol Chem 249(24):8019–8029

  25. Schmid B, Kromer M, Schulz GE (1996) Expression of porin from Rhodopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure. FEBS Lett 381(1–2):111–114

  26. Surrey T, Jahnig F (1992) Refolding and oriented insertion of a membrane protein into a lipid bilayer. Proc Natl Acad Sci U S A 89(16):7457–7461

    CAS  Article  Google Scholar 

  27. Swick MC, Morgan-Linnell SK, Carlson KM, Zechiedrich L (2011) Expression of multidrug efflux pump genes acrAB-tolC, mdfA, and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob Agents Chemother 55(2):921–924

  28. Theunissen S, Vergauwen B, De Smet L, Van Beeumen J, Van Gelder P, Savvides SN (2009) The agglutination protein AggA from Shewanella oneidensis MR-1 is a TolC-like protein and forms active channels in vitro. Biochem Biophys Res Commun 386(2):380–385

  29. Tikhonova EB, Dastidar V, Rybenkov VV, Zgurskaya HI (2009) Kinetic control of TolC recruitment by multidrug efflux complexes. Proc Natl Acad Sci U S A 106(38):16416–16421

    CAS  Article  Google Scholar 

  30. Tikhonova EB, Devroy VK, Lau SY, Zgurskaya HI (2007) Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB. Mol Microbiol 63(3):895–910

  31. Tikhonova EB, Zgurskaya HI (2004) AcrA, AcrB, and TolC of Escherichia coli form a stable intermembrane multidrug efflux complex. J Biol Chem 279(31):32116–32124

  32. Visudtiphole V, Thomas MB, Chalton DA, Lakey JH (2005) Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers. Biochem J 392(Pt 2):375–381

  33. Williams DC, Van Frank RM, Muth WL, Burnett JP (1982) Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science 215(4533):687–689

  34. Wu EL, Fleming PJ, Yeom MS, Widmalm G, Klauda JB, Fleming KG, Im W (2014) Escherichia coli outer membrane and interactions with OmpLA. Biophys J 106(11):2493–2502

    CAS  Article  Google Scholar 

  35. Zakharov SD, Eroukova VY, Rokitskaya TI, Zhalnina MV, Sharma O, Loll PJ, Zgurskaya HI, Antonenko YN, Cramer WA (2004) Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import. Biophys J 87(6):3901–3911

    CAS  Article  Google Scholar 

  36. Zakharov SD, Wang XS, Cramer WA (2016) The colicin E1 TolC-binding conformer: pillar or pore function of TolC in colicin import? Biochemistry 55(36):5084–5094

    CAS  Article  Google Scholar 

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We gratefully acknowledge Rik Dhar, Alex Little, and Jaden Anderson for discussions and feedback, Rajeev Misra for the pTrc vector containing the TolC gene, and Vasileios Petrou for guidance on nanodiscs.


The Gordon and Betty Moore Inventor Fellowship to JSGS and NIGMS award DP2GM128201 to Joanna Slusky, and P20GM103638 and Kansas INBRE, P20 GM103418 and 2K12GM063651 to Jimmy Budiardjo. We are also grateful for inspiration and mentorship from Stephen White. Our understanding of membrane protein folding is richer for your contributions and conferences are more fun when you are there. Thank you for leading the way in asking the important questions. Happy birthday and many happy returns.

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Correspondence to Joanna S. G. Slusky.

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Budiardjo, S.J., Ikujuni, A.P., Firlar, E. et al. High-Yield Preparation of Outer Membrane Protein Efflux Pumps by in Vitro Refolding is Concentration Dependent. J Membrane Biol 254, 41–50 (2021). https://doi.org/10.1007/s00232-020-00161-y

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  • Outer membrane protein
  • In vitro folding
  • Antibiotic resistance