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 [2H,13C,15N]-labeled protein are shown to yield narrow 13C 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.
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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05-20-2016 12:05 AM
[NMR paper] 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.
Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
Protein Expr Purif. 2014 Nov 12;
Authors: Chadwick AC, Jensen DR, Peterson FC, Volkman BF, Sahoo D
Abstract
Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of...
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12-03-2014 04:05 PM
[NMR paper] Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
Protein Expr Purif. 2014 Aug 12;
Authors: Chen W, Cotten ML
Abstract
Piscidin 1 and piscidin 3, which were discovered in the mast cells of hybrid striped sea bass, are homologous antimicrobial peptides that are active against drug-resistant bacteria. Piscidin 1, the more antimicrobial and hemolytic...
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08-19-2014 11:21 AM
[NMR paper] Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay.
Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay.
Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay.
Protein Expr Purif. 2013 Aug 1;
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Abstract
We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled...
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08-07-2013 01:24 PM
Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.
Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.
Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.
Protein Expr Purif. 2011 Aug 31;
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Abstract
Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid ? peptide (A?). A? aggregation produces...
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09-13-2011 08:27 PM
Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements.
Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements.
Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements.
Protein Expr Purif. 2011 May 24;
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Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus...
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06-07-2011 11:05 AM
Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies.
Protein Expr Purif. 2011 Feb 11;
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LR11 (SorLA) is a recently identified neuronal protein that interacts with amyloid precursor protein (APP), a central player...
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02-16-2011 07:40 PM
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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