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Expression and purification of the membrane protein p7 from hepatitis C virus
Author(s) -
Cook Gabriel A.,
Stefer Susanne,
Opella Stanley J.
Publication year - 2011
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.21453
Subject(s) - chemistry , nanodisc , nuclear magnetic resonance spectroscopy , model lipid bilayer , solid state nuclear magnetic resonance , membrane , phosphocholine , membrane protein , heteronuclear single quantum coherence spectroscopy , micelle , crystallography , lipid bilayer , biophysics , phospholipid , biochemistry , stereochemistry , nuclear magnetic resonance , organic chemistry , physics , phosphatidylcholine , aqueous solution , biology , lipid bilayer phase behavior
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. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 32–40, 2011.