Expression and Purification of a Multidrug Transporter PfMATE for Structural Analyses Using Solution NMR

Multidrug and toxin extrusion (MATE) family transporters excrete various toxic compounds coupled to Na⁺/H⁺ influx. In a recent breakthrough, a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution NMR has unveiled how substrate export is coupled to ion influx. This significant advancement was made possible by expressing a stable-isotope-labeled PfMATE in an E. coli in vivo expressing system and an in vitro expression system using a commercially available E. coli-based in vitro expression system. In this chapter, we will delve into the details of both expression systems and provide a comprehensive summary of how stable-isotope-labeled PfMATE was expressed, purified, and subjected to the NMR analyses.