Protein nuclear magnetic resonance*(NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ? subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 ?M from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.
Deuteration and selective labeling of alanine methyl groups of β 2 -adrenergic receptor expressed in a baculovirus-insect cell expression system
Deuteration and selective labeling of alanine methyl groups of β 2 -adrenergic receptor expressed in a baculovirus-insect cell expression system
Abstract
G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited....
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[NMR paper] Sequential protein expression and selective labeling for in-cell NMR in human cells.
Sequential protein expression and selective labeling for in-cell NMR in human cells.
Related Articles Sequential protein expression and selective labeling for in-cell NMR in human cells.
Biochim Biophys Acta. 2015 Dec 23;
Authors: Luchinat E, Secci E, Cencetti F, Bruni P
Abstract
BACKGROUND: In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically...
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Sequential protein expression and selective labeling for in-cell NMR in human cells
Sequential protein expression and selective labeling for in-cell NMR in human cells
Publication date: Available online 23 December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects</br>
Author(s): Enrico Luchinat, Erica Secci, Francesca Cencetti, Paola Bruni</br>
Background In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically labeled. Here we show that...
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12-28-2015 12:26 AM
[NMR paper] Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.
Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.
Related Articles Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.
PLoS One. 2013;8(5):e62947
Authors: Isaksson L, Mayzel M, Saline M, Pedersen A, Rosenlöw J, Brutscher B, Karlsson BG, Orekhov VY
Abstract
We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition,...
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05-15-2013 03:12 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...
Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein
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