Abstract
The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.
Highlights
It has been estimated that membrane proteins account for approximately 20-30% of the genes coded in all sequenced genomes 1, including the human genome 2
In this article we describe a streamlined protocol for cloning and expression screening of membrane proteins in E. coli using fusion to GFP as a reporter of production and subsequent characterization of detergent: protein complexes (Figure 1)
A small scale expression screen of the constructs was carried out in two E .coli strains, Lemo21(DE3) grown in the presence of 0.25 mM rhamnose to block leaky expression and C41(DE3) pLysS, which are routinely used for the expression of membrane proteins[5,6,7,8]
Summary
It has been estimated that membrane proteins account for approximately 20-30% of the genes coded in all sequenced genomes 1, including the human genome 2 Given their critical role in many biological processes, for example transport of metabolites, energy generation and as drug targets, there is intense interest in determining their three dimensional structure. Of the 99,624 released structures in the PDB (http://www.rcsb.org/pdb/) only 471 (http://blanco.biomol.uci.edu/mpstruc/) are classified as membrane proteins. This reflects the technical difficulties of working with membrane proteins which are naturally expressed at relatively low levels; rhodopsin is one of a few exceptions[3,4]. In this article we describe a streamlined protocol for cloning and expression screening of membrane proteins in E. coli using fusion to GFP as a reporter of production and subsequent characterization of detergent: protein complexes (Figure 1)
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