Preparation of Glycerol Facilitator for Protein Structure and Folding Studies in Solution

  • Darren Manley
  • Joe D. O’ Neil
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 228)

Abstract

Progress in the elucidation of protein folding pathways is contributing to a deeper understanding of the information encoded in genomes (1). However, the study of membrane protein folding lags behind similar studies of water-soluble proteins (2). Some of the impediments include poor expression of membrane proteins possibly caused by their misfolding, aggregation, improper transport, and insertion into membranes, or their toxicity when overexpressed (3). In vitro, poor solubility properties lead to difficulties in purification, refolding, crystallization, and nuclear magnetic resonance analysis. The Escherichia coli glycerol facilitator (GF) is an attractive model for studying membrane protein folding (4, 5, 6). E. coli is by far the most successful and versatile vehicle for the production of recombinant proteins and by expressing a native bacterial protein in E. coli difficulties associated with the expression of foreign genes, such as rare codon usage (7) and posttranslational modifications, are avoided. High levels of GF protein are unlikely to be toxic to the cell; the facilitator is highly specific for glycerol transport (5,6) and glycerol can be omitted from the growth medium. Uncontrolled water transport might be problematic owing to osmotic pressure effects, however, the facilitator does not transport water nearly as efficiently as the aquaporins, and aquaporin-1 is naturally highly expressed in red blood cells (5,6). In addition, it seems unlikely that high levels of intracellular glycerol would be toxic to E. coli and in indeed we find maximum expression using the glycerol-rich medium Terrific Broth (TB) (8).

Keywords

Crystallization Glycerol Sedimentation Shrinkage Boiling 

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Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Darren Manley
    • 1
  • Joe D. O’ Neil
    • 1
  1. 1.Department of ChemistryUniversity of ManitobaWinnipegCanada

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