How to isolate channel-forming membrane proteins using the E. coli expression system

Abstract

The recombinant expression, isolation and characterization of pore-forming proteins is one of the most commonly used strategies for understanding the permeability properties of the biological membrane into which they are embedded. This protocol describes how to quantify the expression of your protein of interest and use this information to optimize its production using the Escherichia coli strain BL21Gold(de3)ΔABCF. It explains with a step-by-step approach how to separate the bacterial compartments according to their solubility and how to extract your protein of interest in its native conformation using detergent solutions. Finally, it describes how to improve its purity via ion-exchange chromatography and insert the purified porins into outer membrane vesicles, from which they can be copurified. The protocol is simpler and less empirical than those described for most channel-forming membrane proteins and also provides a solid foundation for the isolation of soluble proteins. Several parameters can be optimized on a case-by-case basis: expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, pH and ionic strength of the purification buffers. This protocol is effective with prokaryotic channel-forming membrane proteins and can be employed for the production of pore-forming proteins from chloroplasts, mitochondria or eukaryotes in general. With minor optimization, this protocol can be adapted for the isolation of receptors, carrier, pumps or any other membrane-active proteins.