Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus
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 sol...
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Veröffentlicht in: | Journal of biomolecular NMR 2016-06, Vol.65 (2), p.87-98 |
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Hauptverfasser: | , , , , , , , , , , , , , |
Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0925-2738 1573-5001 |
DOI: | 10.1007/s10858-016-0040-2 |