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Protein Expr Purif. 2013 Oct;91(2):119-24. doi: 10.1016/j.pep.2013.07.013. Epub 2013 Aug 1.

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

Author information

1
Department of Chemistry, Columbia University, NY 10027, United States. Electronic address: mpb2124@columbia.edu.

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 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.

KEYWORDS:

Isotopic labeling; Liposomes; Membrane proteins; Reconstitution; Solid-state NMR

PMID:
23916531
PMCID:
PMC3805054
DOI:
10.1016/j.pep.2013.07.013
[Indexed for MEDLINE]
Free PMC Article

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