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

Détails

ID Serval
serval:BIB_F87372E38AB4
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
Périodique
Journal of biomolecular NMR
Auteur(s)
Fogeron M.L., Jirasko V., Penzel S., Paul D., Montserret R., Danis C., Lacabanne D., Badillo A., Gouttenoire J., Moradpour D., Bartenschlager R., Penin F., Meier B.H., Böckmann A.
ISSN
1573-5001 (Electronic)
ISSN-L
0925-2738
Statut éditorial
Publié
Date de publication
06/2016
Peer-reviewed
Oui
Volume
65
Numéro
2
Pages
87-98
Langue
anglais
Notes
Publication types: Journal Article
Publication Status: ppublish
Résumé
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.

Mots-clé
Amino Acid Sequence, Carbon-13 Magnetic Resonance Spectroscopy, Circular Dichroism, Gene Expression, Humans, Magnetic Resonance Spectroscopy/methods, Membrane Proteins/chemistry, Nuclear Magnetic Resonance, Biomolecular/methods, Protein Domains, Proteolipids/chemistry, Viral Nonstructural Proteins/chemistry, Viral Nonstructural Proteins/genetics, Viral Nonstructural Proteins/isolation & purification, Cell-free protein expression, Integral membrane protein, Isotope labeling, Lipid reconstitution, NS4B, Solid-state NMR
Pubmed
Web of science
Création de la notice
14/06/2016 17:17
Dernière modification de la notice
20/08/2019 17:24
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