Expression and purification of the membrane protein p7 from hepatitis C virus.
A small 63-residue membrane protein, p7, has essential roles in the infectivity of the hepatitis C virus in humans. This hydrophobic membrane protein forms homo-oligomeric ion channels in bilayers, which can be blocked by known channel-blocking compounds. To perform structural studies of p7 by nuclear magnetic resonance (NMR) spectroscopy, it is necessary to produce milligram quantities of isotopically labeled protein; as is the case for most membrane-associated proteins, this is challenging. We describe the successful expression of full-length p7 and two truncated constructs in Escherichia coli using a fusion partner that directs the overexpressed protein to inclusion bodies. Following isolation of the fusion proteins by affinity chromatography, they were chemically cleaved with cyanogen bromide. The p7-polypeptides were purified by size-exclusion chromatography. Solution NMR two-dimensional heteronuclear single quantum coherence spectra of uniformly (15) N-labeled p7-polypeptides in 1,2-dihexyl-1-sn-glycero-3-phosphocholine isotropic micelles are fully resolved, with a single resonance for each amide site. The solid-state NMR spectra of the same polypeptides in magnetically aligned 14-O-PC/6-O-PC bicelles demonstrate their reconstitution into planar phospholipid bilayers.
Cook
Gabriel A
GA
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0307, USA.
Stefer
Susanne
S
Opella
Stanley J
SJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000086082:Viroporin Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins