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

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 sol...

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Hauptverfasser: Fogeron, Marie-Laure (VerfasserIn) , Jirasko, Vlastimil (VerfasserIn) , Penzel, Susanne (VerfasserIn) , Paul, David (VerfasserIn) , Montserret, Roland (VerfasserIn) , Danis, Clément (VerfasserIn) , Lacabanne, Denis (VerfasserIn) , Badillo, Aurélie (VerfasserIn) , Gouttenoire, Jérôme (VerfasserIn) , Moradpour, Darius (VerfasserIn) , Bartenschlager, Ralf (VerfasserIn) , Penin, François (VerfasserIn) , Meier, Beat H. (VerfasserIn) , Böckmann, Anja (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: 27 May 2016
In: Journal of biomolecular NMR
Year: 2016, Jahrgang: 65, Heft: 2, Pages: 87-98
ISSN:1573-5001
DOI:10.1007/s10858-016-0040-2
Online-Zugang:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1007/s10858-016-0040-2
Volltext
Verfasserangaben:Marie-Laure Fogeron · Vlastimil Jirasko · Susanne Penzel · David Paul · Roland Montserret · Clément Danis · Denis Lacabanne · Aurélie Badillo · Jérôme Gouttenoire · Darius Moradpour · Ralf Bartenschlager · François Penin · Beat H. Meier · Anja Böckmann
Beschreibung
Zusammenfassung: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.
Beschreibung:Gesehen am 20.05.2020
Beschreibung:Online Resource
ISSN:1573-5001
DOI:10.1007/s10858-016-0040-2

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