Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
- Fogeron ML 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.
- Jirasko V Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
- Penzel S Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
- Paul D Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120, Heidelberg, Germany.
- Montserret R 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.
- Danis C 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.
- Lacabanne D 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.
- Badillo A 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.
- Gouttenoire J Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011, Lausanne, Switzerland.
- Moradpour D Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011, Lausanne, Switzerland.
- Bartenschlager R Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120, Heidelberg, Germany.
- Penin F 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.
- Meier BH Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland. beme@ethz.ch.
- Böckmann A 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.
- 2016-05-29
Published in:
- Journal of biomolecular NMR. - 2016
Cell-free protein expression
Integral membrane protein
Isotope labeling
Lipid reconstitution
NS4B
Solid-state NMR
Amino Acid Sequence
Carbon-13 Magnetic Resonance Spectroscopy
Circular Dichroism
Gene Expression
Humans
Magnetic Resonance Spectroscopy
Membrane Proteins
Nuclear Magnetic Resonance, Biomolecular
Protein Domains
Proteolipids
Viral Nonstructural Proteins
English
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.
- Language
-
- English
- Open access status
- green
- Identifiers
-
- DOI 10.1007/s10858-016-0040-2
- PMID 27233794
- Persistent URL
- https://sonar.ch/global/documents/68150
Statistics
Document views: 38
File downloads:
- Full-text: 0