The Expression,\nPurification, Spectroscopic Characterization,\nand Membrane Topology Classification of KCNE4 from Recombinant E. coli

Members of the KCNE family are accessory subunits that\nmodulate\nvoltage-gated potassium channels. One member, KCNE4, has been shown\nto inhibit the potassium ion current in these channels. However, little\nis known about the structure, dynamics, and mode of inhibition of\nKCNE4, likely due to challenges in overexpressing and purifying the\nprotein. In this study, an alternative expression and purification\nprotocol has been developed and validated to obtain overexpressed\nKCNE4 for <i>in vitro</i> studies. This protocol was validated\nthrough SDS-PAGE, CW-EPR, CW-EPR power saturation, and CD experiments.\nThe SDS-PAGE and CD data reveal that this protocol produces relatively\npure and properly folded KCNE4 in large quantities at a lower cost.\nThe CW-EPR and EPR power saturation spectra show that KCNE4 consists\nof extracellular, transmembrane, and intracellular regions. Together,\nthese techniques indicate that this alternative protocol produces\nstructurally and dynamically native KCNE4 without the need for mammalian\ncell lines. This study provides guidance for characterizing the structure\nand dynamics of KCNE4 in a lipid bilayer environment.