Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay

We report the expression, purification, liposome reconstitution and functional validation of uniformly C-13 and N-15 isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of similar to 35-40 mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR. (C) 2013 Elsevier Inc. All rights reserved.