PMID:20196076
| Citation |
Hsieh, JM, Besserer, GM, Madej, MG, Bui, HQ, Kwon, S and Abramson, J (2010) Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini. Protein Sci. 19:868-80 |
|---|---|
| Abstract |
Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C(in)) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C(out)) to C(in) ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C(out) topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C(out) topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging. |
| Links |
PubMed PMC2867025 Online version:10.1002/pro.365 |
| Keywords |
Chromatography, Gel; Escherichia coli/genetics; Escherichia coli/metabolism; Genetic Vectors/genetics; Genetic Vectors/metabolism; Green Fluorescent Proteins/analysis; Green Fluorescent Proteins/genetics; Membrane Proteins/genetics; Membrane Proteins/isolation & purification; Membrane Proteins/metabolism; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism |
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