The rapid "teabag" method for high-end purification of membrane proteins.

Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast "teabag" purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences.

Membrane Protein Production in E. coli for Applications in Drug Discovery.

Producing high quality purified membrane proteins for structure-based drug design and biophysical assays compatible with typical timelines in drug discovery is a significant challenge. Escherichia coli has been an expression host of the utmost importance for soluble proteins and has applications for membrane proteins as well. However, membrane protein overexpression in E. coli may lead to toxicity and low yields of functional product. Here, we review the challenges encountered with heterologous overproduction of α-helical membrane proteins in E. coli and a range of strategies to overcome them. A detailed protocol is also provided for expression and screening of membrane proteins in E. coli using a His-specific fluorescent probe and fluorescent size-exclusion chromatography.