Microscale Thermophoresis to Evaluate the Functionality of Heterologously Overexpressed Membrane Proteins in Membrane Preparations

Transmembrane proteins are challenging to express in heterologous systems and to purify, thus any technique enabling to evaluate the functionality of the protein produced prior purification provides a huge step forward. Furthermore, the membrane environment may be critical for the activity of the target protein and accessing information in the membrane fragments instead of solubilizing the target into a detergent that may be unsuitable for its function is key to study and evaluate its activity. Herein, we describe how microscale thermophoresis (MST) was used to evaluate the functionality of membrane proteins directly in host membrane preparation before purification. We give a protocol to measure the affinity between the human Hedgehog (Hh) receptor Ptch1 in yeast plasma membrane and the small molecule PAH, which was shown to inhibit its drug efflux activity.