Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
J Biomol NMR. 2016 May 27;
Authors: Fogeron ML, Jirasko V, Penzel S, Paul D, Montserret R, Danis C, Lacabanne D, Badillo A, Gouttenoire J, Moradpour D, Bartenschlager R, Penin F, Meier BH, Böckmann A
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 ?-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.
PMID: 27233794 [PubMed - as supplied by publisher]
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus
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 -labeled protein are shown to yield narrow 13C...
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Kinetic, Mutational, and Structural Studies of theVenezuelan Equine Encephalitis Virus Nonstructural Protein 2 CysteineProtease
Kinetic, Mutational, and Structural Studies of theVenezuelan Equine Encephalitis Virus Nonstructural Protein 2 CysteineProtease
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/0/bichaw.ahead-of-print/acs.biochem.5b00992/20160519/images/medium/bi-2015-00992a_0007.gif
Biochemistry
DOI: 10.1021/acs.biochem.5b00992
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Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
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Authors: Chadwick AC, Jensen DR, Peterson FC, Volkman BF, Sahoo D
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Authors: Prchal J, Junkova P, Strmiskova M, Lipov J, Hynek R, Ruml T, Hrabal R
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Authors: Wang X, Gill Jr RL, Zhu Q, Tian F
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Cell-free expression and stable isotope labelling strategies for membrane proteins
Cell-free expression and stable isotope labelling strategies for membrane proteins
Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
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