Optimization of membrane protein overexpression and purification using GFP fusions

Optimizing conditions for the overexpression and purification of membrane proteins for functional and structural studies is usually a laborious and time-consuming process. This process can be accelerated using membrane protein–GFP fusions 1–3 , which allows direct monitoring and visualization of membrane proteins of interest at any stage during overexpression, solubilization and purification (Fig. 1). The exceptionally stable GFP moiety of the fusion protein can be used to detect membrane proteins by observing fluorescence in whole cells during overexpression, with a detection limit as low as 10 µg of GFP per liter of culture, and in solution during solubilization and purification. Notably, the fluorescence of the GFP moiety can also be detected in standard SDS polyacrylamide gels with a detection limit of less than 5 ng of GFP per protein band (Fig. 2). In-gel fluorescence allows assessment of the integrity of membrane protein–GFP fusions and provides a rapid and generic alternative for the notoriously difficult immunoblotting of membrane proteins. With whole-cell and in-gel fluorescence the overexpression potential of many membrane protein–GFP fusions can be rapidly assessed and yields of promising targets can be improved. In this protocol the Escherichia coli BL21(DE3)-pET system—the most widely used (membrane) protein overexpression system—is used as a platform to illustrate the GFP-based method. The methodology described in this protocol can be transferred easily to other systems.