We are seeing patients in-person and through Video Visits. Learn more about how we’re keeping you safe and please review our updated visitor policy. Please also consider supporting Weill Cornell Medicine’s efforts to support our front-line workers.
Department of Anesthesiology

You are here

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

TitlePreparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.
Publication TypeJournal Article
Year of Publication2013
AuthorsBhate MP, Wylie BJ, Thompson A, Tian L, Nimigean C, McDermott AE
JournalProtein Expr Purif
Volume91
Issue2
Pagination119-24
Date Published2013 Oct
ISSN1096-0279
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.

DOI10.1016/j.pep.2013.07.013
Alternate JournalProtein Expr. Purif.
PubMed ID23916531
PubMed Central IDPMC3805054
Grant ListF32 087908 / / PHS HHS / United States
P41GM66354 / GM / NIGMS NIH HHS / United States
R01 GM088724 / GM / NIGMS NIH HHS / United States
R01 GM88724 / GM / NIGMS NIH HHS / United States
R01GM088352 / GM / NIGMS NIH HHS / United States