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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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Main Authors: Fogeron, Marie-Laure (Author) , Jirasko, Vlastimil (Author) , Penzel, Susanne (Author) , Paul, David (Author) , Montserret, Roland (Author) , Danis, Clément (Author) , Lacabanne, Denis (Author) , Badillo, Aurélie (Author) , Gouttenoire, Jérôme (Author) , Moradpour, Darius (Author) , Bartenschlager, Ralf (Author) , Penin, François (Author) , Meier, Beat H. (Author) , Böckmann, Anja (Author)
Format: Article (Journal)
Language:English
Published: 27 May 2016
In: Journal of biomolecular NMR
Year: 2016, Volume: 65, Issue: 2, Pages: 87-98
ISSN:1573-5001
DOI:10.1007/s10858-016-0040-2
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1007/s10858-016-0040-2
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Author Notes: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
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Summary: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.
Item Description:Gesehen am 20.05.2020
Physical Description:Online Resource
ISSN:1573-5001
DOI:10.1007/s10858-016-0040-2

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