Refolding of a membrane protein in a microfluidics reactor

Eur Biophys J. 2007 Jul;36(6):581-8. doi: 10.1007/s00249-006-0125-z. Epub 2007 Jan 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriorhodopsins / chemistry
  • Dimyristoylphosphatidylcholine / chemistry
  • Green Fluorescent Proteins / chemistry
  • Micelles
  • Microfluidics / instrumentation
  • Microfluidics / methods*
  • Protein Folding*

Substances

  • Micelles
  • Green Fluorescent Proteins
  • Bacteriorhodopsins
  • Dimyristoylphosphatidylcholine