Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus
Abstract
We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [2H,13C,15N]-labeled protein are shown to yield narrow 13C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000118305Publication status
publishedExternal links
Journal / series
Journal of Biomolecular NMRVolume
Pages / Article No.
Publisher
SpringerSubject
Cell-free protein expression; Integral membrane protein; Isotope labeling; Lipid reconstitution; NS4B; Solid-state NMROrganisational unit
03496 - Meier, Beat H. (emeritus) / Meier, Beat H. (emeritus)
Funding
146757 - NMR studies in the Solid State (SNF)
159707 - NMR studies in the Solid State (SNF)
Notes
It was possible to publish this article open access thanks to a Swiss National Licence with the publisher.More
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