Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

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Fogeron, Marie-Laure | Jirasko, Vlastimil | Penzel, Susanne | Paul, David | Montserret, Roland | Danis, Clément | Lacabanne, Denis | Badillo, Aurélie | Gouttenoire, Jérôme | Moradpour, Darius | Bartenschlager, Ralf | Penin, François | Meier, Beat | Böckmann, Anja

Edité par CCSD ; Springer Verlag -

International audience. 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 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.

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