Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.
[NMR paper] Cost-effective large-scale expression of proteins for NMR studies.
Cost-effective large-scale expression of proteins for NMR studies.
Related Articles Cost-effective large-scale expression of proteins for NMR studies.
J Biomol NMR. 2018 May 19;:
Authors: Klopp J, Winterhalter A, Gébleux R, Scherer-Becker D, Ostermeier C, Gossert AD
Abstract
We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13C-methyl methionine labeling in rich media, where yields of up to a gram of...
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05-21-2018 06:16 PM
Cost-effective large-scale expression of proteins for NMR studies
Cost-effective large-scale expression of proteins for NMR studies
Abstract
We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2H, 13C and 15N using auto-induction or isopropyl-β-d-1-thiogalactopyranoside-induction are described, with primary focus on...
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05-19-2018 05:41 PM
[NMR paper] Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.
J Struct Biol. 2014 Aug 27;
Authors: Meola A, Deville C, Jeffers SA, Guardado-Calvo P, Vasiliauskaite I, Sizun C, Girard-Blanc C, Malosse C, Heijenoort CV, Chamot-Rooke J, Krey T, Guittet E, Pêtres S, Rey FA, Bontems F
Abstract
Nuclear...
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09-01-2014 07:46 PM
Robust and low cost uniform 15N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
Robust and low cost uniform 15N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
Publication date: Available online 28 August 2014
Source:Journal of Structural Biology</br>
Author(s): Annalisa Meola , Célia Deville , Scott A. Jeffers , Pablo Guardado-Calvo , Ieva Vasiliauskaite , Christina Sizun , Christine Girard-Blanc , Christian Malosse , Carine van Heijenoort , Julia Chamot-Rooke , Thomas Krey , Eric Guittet , Stéphane Pêtres , Félix A. Rey , François Bontems</br>
Nuclear magnetic...
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08-29-2014 05:36 PM
[NMR paper] Effective strategy to assign (1)H- (15)N heteronuclear correlation NMR signals from lysine side-chain NH3 (+) groups of proteins at low temperature.
Effective strategy to assign (1)H- (15)N heteronuclear correlation NMR signals from lysine side-chain NH3 (+) groups of proteins at low temperature.
Related Articles Effective strategy to assign (1)H- (15)N heteronuclear correlation NMR signals from lysine side-chain NH3 (+) groups of proteins at low temperature.
J Biomol NMR. 2014 Aug 17;
Authors: Esadze A, Zandarashvili L, Iwahara J
Abstract
Recent studies have shown that lysine side-chain NH3 (+) groups are excellent probes for NMR investigations of dynamics involving...
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08-19-2014 11:21 AM
Effective strategy to assign 1H-15N heteronuclear correlation NMR signals from lysine side-chain NH3 + groups of proteins at low temperature
Effective strategy to assign 1H-15N heteronuclear correlation NMR signals from lysine side-chain NH3 + groups of proteins at low temperature
Abstract
Recent studies have shown that lysine side-chain NH3 + groups are excellent probes for NMR investigations of dynamics involving hydrogen bonds and ion pairs relevant to protein function. However, due to rapid hydrogen exchange, observation of 1H-15N NMR cross peaks from lysine NH3 + groups often requires use of a relatively low temperature, which...
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08-16-2014 10:26 PM
[NMR paper] Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
Related Articles Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
Protein Expr Purif. 2014 Mar 21;
Authors: Kroupa T, Prchal J, Doležal M, Ruml T, Hrabal R
Abstract
Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures orstudying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon(13)C and...
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03-26-2014 12:44 PM
A cost-effective look at proteins - with a little help from robotics - Cordis News
http://www.bionmr.com//t3.gstatic.com/images?q=tbn:ANd9GcTypMhbcsoTBmkGzm1ZmzjTHj-b-AsNVL4y8L03Y0oBfj2cCZUDv2gZmGEzmim-0VUKjKStcwU
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A cost-effective look at proteins - with a little help from robotics
Cordis News
Until now, a number of methods have been used to study static protein structures, including X-ray crystallography and nuclear magnetic resonance. However, these methods are of no use for proteins that are on the move; analytical methods, computer ...
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A cost-effective look at proteins - with a little help from...
Towards cost-effective side-chain isotope labelling of proteins expressed in human cells
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