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

Details

Serval ID
serval:BIB_F87372E38AB4
Type
Article: article from journal or magazin.
Collection
Publications
Title
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
Journal
Journal of biomolecular NMR
Author(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
Publication state
Published
Issued date
06/2016
Peer-reviewed
Oui
Volume
65
Number
2
Pages
87-98
Language
english
Notes
Publication types: Journal Article
Publication Status: ppublish
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.

Keywords
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
Create date
14/06/2016 17:17
Last modification date
20/08/2019 17:24
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