European Biophysics Journal

, Volume 36, Issue 6, pp 581–588 | Cite as

Refolding of a membrane protein in a microfluidics reactor

  • Nathan R. ZaccaiEmail author
  • Kamran Yunus
  • S. M. Matthews
  • Adrian C. Fisher
  • Robert J. Falconer
Original Paper


Membrane protein production for structural studies is often hindered by the formation of non-specific aggregates from which the protein has to be denatured and then refolded to a functional state. We developed a new approach, which uses microfluidics channels, to refold protein correctly in quantities sufficient for structural studies. Green fluorescent protein (GFP), a soluble protein, and bacteriorhodopsin (BR), a transmembrane protein, were used to demonstrate the efficiency of the process. Urea-denatured GFP refolded as the urea diffused away from the protein, forming in the channel a uniform fluorescent band when observed by confocal microscopy. Sodium dodecyl sulphate-denatured BR refolded within the channel on mixing with detergent–lipid mixed micelles. The refolding, monitored by absorbance spectroscopy, was found to be flow rate dependent. This potential of microfluidic reactors for screening protein-folding conditions and producing protein would be particularly amenable for high-throughput applications required in structural genomics.


Green fluorescent protein Bacteriorhodopsin Microfluidics reactor Membrane protein refolding Structural genomics 













Green fluorescent protein


Glutathione S-transferase


Sodium dodecyl sulphate





This work was supported by the Biotechnology and Biological Sciences Research Council (UK). We thank A. Domin for the GFP plasmid, G. Zaccai for purple membrane from H. salinarium and J. Skepper for the help with confocal microscopy. We are particularly grateful to I. Falconer, S. Hanslip, M. Hutchinson and G. Zaccai for their insightful comments.

Supplementary material


  1. Akiyama S, Takahashi S, Ishimori K, Morishima I (2000) Stepwise formation of alpha-helices during cytochrome c folding. Nat Struct Biol 7:514–520CrossRefGoogle Scholar
  2. Andersson A, Maler L (2005) Magnetic resonance investigations of lipid motion in isotropic bicelles. Langmuir 21:7702–7709CrossRefGoogle Scholar
  3. Armstrong N, de Lencastre A, Gouaux E (1999) A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci 8:1475–1483Google Scholar
  4. Baneres JL, Martin A, Hullot P, Girard JP, Rossi JC, Parello J (2003) Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1. J Mol Biol 329:801–814CrossRefGoogle Scholar
  5. Booth PJ (2003) The trials and tribulations of membrane protein folding in vitro. Biochim Biophys Acta 1610:51–56CrossRefGoogle Scholar
  6. Booth PJ, Farooq A, Flitsch SL (1996) Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin. Biochemistry 35:5902–5909CrossRefGoogle Scholar
  7. Christendat D, Yee A, Dharamsi A, Kluger Y, Savchenko A, Cort JR, Booth V, Mackereth CD, Saridakis V, Ekiel I, Kozlov G, Maxwell KL, Wu N, McIntosh LP, Gehring K, Kennedy MA, Davidson AR, Pai EF, Gerstein M, Edwards AM, Arrowsmith CH (2000) Structural proteomics of an archaeon. Nat Struct Biol 7:903–909CrossRefGoogle Scholar
  8. Cody CW, Prasher DC, Westler WM, Prendergast FG, Ward WW (1993) Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry 32:1212–1218CrossRefGoogle Scholar
  9. Dayel MJ, Hom EF, Verkman AS (1999) Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum. Biophys J 76:2843–2851CrossRefGoogle Scholar
  10. Einstein A (1905) Uber die von der molekularkinetischen Theorie der Wärme gefordete Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen. Ann Phys 17:549–560CrossRefGoogle Scholar
  11. Engel A, Muller DJ (2000) Observing single biomolecules at work with the atomic force microscope. Nat Struct Biol 7:715–718CrossRefGoogle Scholar
  12. Enoki S, Saeki K, Maki K, Kuwajima K (2004) Acid denaturation and refolding of green fluorescent protein. Biochemistry 43:14238–14248CrossRefGoogle Scholar
  13. Fukuda H, Arai M, Kuwajima K (2000) Folding of green fluorescent protein and the cycle3 mutant. Biochemistry 39:12025–12032CrossRefGoogle Scholar
  14. Gabel F, Bicout D, Lehnert U, Tehei M, Weik M, Zaccai G (2002) Protein dynamics studied by neutron scattering. Q Rev Biophys 35:327–367CrossRefGoogle Scholar
  15. Geppert L (1996) Semiconductor lithography for the next millennium. IEEE Spectr 33:33–38CrossRefGoogle Scholar
  16. Gordeliy VI, Labahn J, Moukhametzianov R, Efremov R, Granzin J, Schlesinger R, Buldt G, Savopol T, Scheidig AJ, Klare JP, Engelhard M (2002) Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Nature 419:484–487CrossRefADSGoogle Scholar
  17. Gosting LJ, Akeley DF (1952) A study of the diffusion of urea in water at 25-degrees with the Gouy interference method. J Am Chem Soc 74:2058–2060CrossRefGoogle Scholar
  18. Greenhalgh DA, Farrens DL, Subramaniam S, Khorana HG (1993) Hydrophobic amino-acids in the retinal-binding pocket of bacteriorhodopsin. J Biol Chem 268:20305–20311Google Scholar
  19. Haupts U, Tittor J, Oesterhelt D (1999) Closing in on bacteriorhodopsin: progress in understanding the molecule. Annu Rev Biophys Biomol Struct 28:367–399CrossRefGoogle Scholar
  20. Hertzog DE, Michalet X, Jager M, Kong X, Santiago JG, Weiss S, Bakajin O (2004) Femtomole mixer for microsecond kinetic studies of protein folding. Anal Chem 76:7169–7178CrossRefGoogle Scholar
  21. Jeong HJ, Markle DA, Owen G, Pease F, Grenville A, Vonbunau R (1994) The future of optical lithography. Solid State Technol 37:39–47Google Scholar
  22. Levenson MD (1995) Extending optical lithography to the gigabit era. Solid State Technol 38:57–66Google Scholar
  23. Morise H, Shimomura O, Johnson FH, Winant J (1974) Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13:2656–2662CrossRefGoogle Scholar
  24. Nguyen NT, Wu ZG (2005) Micromixers—a review. J Micromech Microeng 15:R1–R16CrossRefGoogle Scholar
  25. Nyquist RM, Ataka K, Heberle J (2004) The molecular mechanism of membrane proteins probed by evanescent infrared waves. Chembiochem 5:431–436CrossRefGoogle Scholar
  26. Oesterhelt D, Stoeckenius W (1974) Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol 31:667–678CrossRefGoogle Scholar
  27. Okazaki S (1991) Resolution limits of optical lithography. J Vac Sci Technol B 9:2829–2833CrossRefGoogle Scholar
  28. Orfi L, Lin MF, Larive CK (1998) Measurement of SDS micelle-peptide association using H-1 NMR chemical shift analysis and pulsed field gradient NMR spectroscopy. Anal Chem 70:1339–1345CrossRefGoogle Scholar
  29. Raman P, Cherezov V, Caffrey M (2006) The membrane protein data bank. Cell Mol Life Sci 63:36–51CrossRefGoogle Scholar
  30. Reid BG, Flynn GC (1997) Chromophore formation in green fluorescent protein. Biochemistry 36:6786–6791CrossRefGoogle Scholar
  31. Scheich C, Niesen FH, Seckler R, Bussow K (2004) An automated in vitro protein folding screen applied to a human dynactin subunit. Protein Sci 13:370–380CrossRefGoogle Scholar
  32. Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666:105–117CrossRefGoogle Scholar
  33. Shastry MC, Luck SD, Roder H (1998) A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale. Biophys J 74:2714–2721Google Scholar
  34. Stevens RC (2000) Design of high-throughput methods of protein production for structural biology. Structure 8:R177–R185CrossRefGoogle Scholar
  35. Subramaniam S, Hirai T, Henderson R (2002) From structure to mechanism: electron crystallographic studies of bacteriorhodopsin. Philos Trans R Soc Lond A Math Phys Eng Sci 360:859–874CrossRefADSGoogle Scholar
  36. Sudarsan AP, Ugaz VM (2006) Fluid mixing in planar spiral microchannels. Lab Chip 6:74–82CrossRefGoogle Scholar
  37. Sugiyama Y, Mukohata Y (1996) Dual roles of DMPC and CHAPS in the refolding of bacterial opsins in vitro. J Biochem 119:1143–1149Google Scholar
  38. Tresaugues L, Collinet B, Minard P, Henckes G, Aufrere R, Blondeau K, Liger D, Zhou CZ, Janin J, Van Tilbeurgh H, Quevillon-Cheruel S (2004) Refolding strategies from inclusion bodies in a structural genomics project. J Struct Funct Genomics 5:195–204CrossRefGoogle Scholar
  39. Waldo GS, Standish BM, Berendzen J, Terwilliger TC (1999) Rapid protein-folding assay using green fluorescent protein [see comment]. Nat Biotechnol 17:691–695CrossRefGoogle Scholar
  40. Watts A (2005) Solid-state NMR in drug design and discovery for membrane-embedded targets. Nat Rev Drug Discov 4:555–568CrossRefGoogle Scholar
  41. Wu T, Mei Y, Cabral JT, Xu C, Beers KL (2004) A new synthetic method for controlled polymerization using a microfluidic system. J Am Chem Soc 126:9880–9881CrossRefGoogle Scholar

Copyright information

© EBSA 2007

Authors and Affiliations

  • Nathan R. Zaccai
    • 2
    Email author
  • Kamran Yunus
    • 1
  • S. M. Matthews
    • 1
  • Adrian C. Fisher
    • 1
  • Robert J. Falconer
    • 3
  1. 1.Department of Chemical EngineeringUniversity of CambridgeCambridgeUK
  2. 2.Department of PharmacologyUniversity of BristolBristolUK
  3. 3.Division of Chemical Engineering, Centre for Biomolecular EngineeringThe University of QueenslandSt LuciaAustralia

Personalised recommendations