Recent Advances in Expression Screening and Sample Evaluation for Structural Studies of Membrane Proteins.

Membrane proteins are involved in numerous biological processes and represent more than half of all drug targets; thus, structural information on these proteins is invaluable. However, the low expression level of membrane proteins, as well as their poor stability in solution and tendency to precipitate and aggregate, are major bottlenecks in the preparation of purified membrane proteins for structural studies. Traditionally, the evaluation of membrane protein constructs for structural studies has been quite time consuming and expensive since it is necessary to express and purify the proteins on a large scale, particularly for X-ray crystallography. The emergence of fluorescence detection size exclusion chromatography (FSEC) has drastically changed this situation, as this method can be used to rapidly evaluate the expression and behavior of membrane proteins on a small scale without the need for purification. FSEC has become the most widely used method for the screening of expression conditions and sample evaluation for membrane proteins, leading to the successful determination of numerous structures. Even in the era of cryo-EM, FSEC and the new generation of FSEC derivative methods are being widely used in various manners to facilitate structural analysis. In addition, the application of FSEC is not limited to structural analysis; this method is also widely used for functional analysis of membrane proteins, including for analysis of oligomerization state, screening of antibodies and ligands, and affinity profiling. This review presents the latest advances and applications in membrane protein expression screening and sample evaluation, with a particular focus on FSEC methods.