title: Refolding of a membrane protein in a microfluidics reactor.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Bristol, University Walk, Bristol, UK. nathan.zaccai@bristol.ac.uk
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Zaccai
authors.forename: Kamran
authors.initials: K
authors.lastname: Yunus
authors.forename: S M
authors.initials: SM
authors.lastname: Matthews
authors.forename: Adrian C
authors.initials: AC
authors.lastname: Fisher
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Falconer
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008823:Micelles; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D017510:Protein Folding
chemical_list: D008823:Micelles; D049452:Green Fluorescent Proteins; D001436:Bacteriorhodopsins; D004134:Dimyristoylphosphatidylcholine