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

ABSTRACT: We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N 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 ∼35-40mg/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 the

SUBMITTER: Bhate MP 

PROVIDER: S-EPMC3805054 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature