Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus
Abstract
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 -labeled protein are shown to yield narrow 13C...
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05-29-2016 11:26 AM
[NMR paper] Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.
J Biomol NMR. 2016 May 27;
Authors: Fogeron ML, Jirasko V, Penzel S, Paul D, Montserret R, Danis C, Lacabanne D, Badillo A, Gouttenoire J, Moradpour D, Bartenschlager R, Penin F, Meier BH, Böckmann A
Abstract
We describe the expression of the hepatitis C...
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05-29-2016 11:26 AM
[NMR paper] Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Methods Mol Biol. 2014;1118:169-87
Authors: Ozawa K, Qi R
Abstract
Cell-free protein synthesis (CFPS) offers a fast and inexpensive approach to selectively label proteins with isotopes that can then be detected by nuclear magnetic resonance (NMR) spectroscopy directly in the translation mixture. We describe...
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01-08-2014 11:23 AM
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Abstract Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that...
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02-16-2011 09:34 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...
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01-09-2011 12:46 PM
[NMR paper] Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
Related Articles Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions.
J Biomol NMR. 2005 Jul;32(3):235-41
Authors: Ozawa K, Jergic S, Crowther JA, Thompson PR, Wijffels G, Otting G, Dixon NA
Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein-protein complexes even if one of the protein components is prone...
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12-01-2010 06:56 PM
[NMR paper] Quantitation of protein expression in a cell-free system: Efficient detection of yiel
Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.
Related Articles Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.
J Biomol NMR. 2005 Jan;31(1):11-9
Authors: Neerathilingam M, Greene LH, Colebrooke SA, Campbell ID, Staunton D
We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a...
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11-24-2010 11:14 PM
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Touraj Etezady-Esfarjani, Sebastian Hiller, Cristina Villalba and Kurt Wüthrich
Journal of Biomolecular NMR; 2007; 39(3); pp 229-238
Abstract:
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins...
Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression
Linear Mode
