Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
J Biomol NMR. 2016 May 27;
Authors: Fogeron ML, Jirasko V, Penzel S, Paul D, Montserret R, Danis C, Lacabanne D, Badillo A, Gouttenoire J, Moradpour D, Bartenschlager R, Penin F, Meier BH, Böckmann A
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 [(2)H,(13)C,(15)N]-labeled protein are shown to yield narrow (13)C resonance lines and a proper, predominantly ?-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.
PMID: 27233794 [PubMed - as supplied by publisher]
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