Determination of membrane protein structures remains exceptionally challenging. One of the central issues is the development of systems that permit the high-level expression of properly folded membrane proteins. To address this issue, part of our efforts focus on developing technologies for the overexpression of membrane proteins for structural study and solving their structures. We have developed facile green fluorescence protein (GFP) based screen for identifying membrane proteins that can be expressed at high level and that yield samples suitable for structural studies. Implementation of this screen to survey the expression properties of >300 membrane protein from extremeophile organisms (Hammon et al., Protein Science, 2009), organisms expected to be rich sources of membrane proteins having robust biophysical properties, has yielded a large pool of well-expressed candidates. We find that GFP-fusion fluorescence intensity is an excellent indicator of over-expression potential. By employing a follow-up optimization protocol using a suite of non-GFP constructs and different expression temperatures, we obtain 0.5-15 mg L-1 expression levels for 90% of candidate proteins that pass the GFP screen. Evaluation of the results suggests that certain organisms may serve as better sources of well-expressed membrane proteins than others, that the degree to which codon usage matches the expression host is uncorrelated with success rate, and that the combination of GFP screening and expression optimization is essential for producing biochemically tractable quantities of material. As a result of our expression screening, we presently have a number of well-expressed candidates that yield monodisperse samples in a variety of detergents. Efforts to determine crystallize and determine the structures of extremophilic membrane proteins are ongoing.

[1] Structural Understanding of Ion Channel Action and Regulation
[2] Molecular Evolution of Ion Channel Modulators for Channels
[3] Membrane Protein Expression and Structure