Refolding of a membrane protein in a microfluidics reactor
- 194 Downloads
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.
KeywordsGreen fluorescent protein Bacteriorhodopsin Microfluidics reactor Membrane protein refolding Structural genomics
Green fluorescent protein
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.
- 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
- 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
- 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
- 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
- Levenson MD (1995) Extending optical lithography to the gigabit era. Solid State Technol 38:57–66Google Scholar
- 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
- 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