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.
%0 Journal Article
%1 cook_expression_2011
%A Cook, Gabriel A
%A Stefer, Susanne
%A Opella, Stanley J
%D 2011
%J Biopolymers
%K Acid Affinity,Amino Data,Polyacrylamide Expression,Magnetic Gel,Recombinant Proteins Proteins,Viral Resonance Sequence Sequence,Base Sequence,Chromatography,Electrophoresis,Escherichia Spectroscopy,Molecular coli,Gel,Gene
%N 1
%P 32--40
%R 10.1002/bip.21453
%T Expression and purification of the membrane protein p7 from hepatitis \C\ virus
%V 96
%X 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.
@article{cook_expression_2011,
abstract = {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.},
added-at = {2017-03-14T02:48:56.000+0100},
author = {Cook, Gabriel A and Stefer, Susanne and Opella, Stanley J},
biburl = {https://www.bibsonomy.org/bibtex/216a2f08c8fc0e4e445aaffdad5051bfd/nmrresource},
doi = {10.1002/bip.21453},
interhash = {d680aa25467788b780c111be7f41b41b},
intrahash = {16a2f08c8fc0e4e445aaffdad5051bfd},
issn = {0006-3525},
journal = {Biopolymers},
keywords = {Acid Affinity,Amino Data,Polyacrylamide Expression,Magnetic Gel,Recombinant Proteins Proteins,Viral Resonance Sequence Sequence,Base Sequence,Chromatography,Electrophoresis,Escherichia Spectroscopy,Molecular coli,Gel,Gene},
number = 1,
pages = {32--40},
pmid = {20560141},
timestamp = {2017-03-14T02:49:21.000+0100},
title = {{Expression and purification of the membrane protein p7 from hepatitis {\{}C{\}} virus}},
volume = 96,
year = 2011
}