title: 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 [(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.
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Fogeron
authors.affiliation: Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.|Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120, Heidelberg, Germany.|German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Jirasko
authors.affiliation: Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
authors.forename: Susanne
authors.initials: S
authors.lastname: Penzel
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120, Heidelberg, Germany.|German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Paul
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.
authors.forename: Roland
authors.initials: R
authors.lastname: Montserret
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.
authors.forename: Clément
authors.initials: C
authors.lastname: Danis
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Lacabanne
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.|Recombinant Protein Unit, RD-Biotech, 3 rue Henri Baigue, 25000, Besançon, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Badillo
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011, Lausanne, Switzerland.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gouttenoire
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011, Lausanne, Switzerland.
authors.forename: Darius
authors.initials: D
authors.lastname: Moradpour
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120, Heidelberg, Germany.|German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland. beme@ethz.ch.
authors.forename: Beat H
authors.identifier: 0000-0002-9107-4464
authors.initials: BH
authors.lastname: Meier
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France. a.bockmann@ibcp.fr.
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
mesh_terms: D000595:Amino Acid Sequence; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D002942:Circular Dichroism; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D000072417:Protein Domains; D011510:Proteolipids / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D008565:Membrane Proteins; C080107:NS4B protein, flavivirus; D011510:Proteolipids; D017361:Viral Nonstructural Proteins; C020485:proteoliposomes
keywords: Cell-free protein expression; Integral membrane protein; Isotope labeling; Lipid reconstitution; NS4B; Solid-state NMR