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

• Published in: Protein expression and purification 2013-10, Vol.91 (2), p.119-124
• Main Authors: Bhate, Manasi P., Wylie, Benjamin J., Thompson, Ameer, Tian, Lin, Nimigean, Crina, McDermott, Ann E.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Isotope Labeling; Isotopic labeling; Liposomes; Liposomes - chemistry; Liposomes - metabolism; Membrane proteins; Nuclear Magnetic Resonance, Biomolecular; Potassium Channels - chemistry; Potassium Channels - genetics; Potassium Channels - isolation & purification; Potassium Channels - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Reconstitution; Solid-state NMR
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2013.07.013

•Protocols to express 40mg/L of pure 15N and 13C labeled KcsA, a bacterial K+ channel for NMR studies are reported.•Optimized protocols to reconstitute membrane proteins into liposomes for solid-state NMR are shown.•Cryo-electron microscopy of liposomes and single-channel measurements of KcsA validate channel function.•These methods yield reliable samples for high-resolution NMR spectra of KcsA for biophysical studies. We report the expression, purification, liposome reconstitution and functional validation of uniformly 13C and 15N 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 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.