The Expression, Purification, Spectroscopic Characterization, and Membrane Topology Classification of KCNE4 from Recombinant <i>E. coli</i>

Members of the KCNE family are accessory subunits that modulate voltage-gated potassium channels. One member, KCNE4, has been shown to inhibit the potassium ion current in these channels. However, little is known about the structure, dynamics, and mode of inhibition of KCNE4, likely due to challenges in overexpressing and purifying the protein. In this study, an alternative expression and purification protocol has been developed and validated to obtain overexpressed KCNE4 for <i>in vitro</i> studies. This protocol was validated through SDS-PAGE, CW-EPR, CW-EPR power saturation, and CD experiments. The SDS-PAGE and CD data reveal that this protocol produces relatively pure and properly folded KCNE4 in large quantities at a lower cost. The CW-EPR and EPR power saturation spectra show that KCNE4 consists of extracellular, transmembrane, and intracellular regions. Together, these techniques indicate that this alternative protocol produces structurally and dynamically native KCNE4 without the need for mammalian cell lines. This study provides guidance for characterizing the structure and dynamics of KCNE4 in a lipid bilayer environment.