Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labeling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labeled substrates. Stable isotope labeling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational...
Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system
Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system
Abstract
Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris)...
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06-05-2018 01:31 PM
[NMR paper] Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Related Articles Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
J Biomol NMR. 2018 Feb 28;:
Authors: Yanaka S, Yagi H, Yogo R, Yagi-Utsumi M, Kato K
Abstract
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing...
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03-02-2018 03:20 PM
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Abstract
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles,...
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02-28-2018 03:32 PM
Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility
Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility
Abstract A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS2) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using...
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03-08-2012 08:46 AM
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 synthesis and amino acid-selective stable isotope labeling of proteins for
Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
Related Articles Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
J Biomol NMR. 1995 Sep;6(2):129-34
Authors: Kigawa T, Muto Y, Yokoyama S
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...
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08-22-2010 03:50 AM
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka
Journal of Biomolecular NMR; 2008; 42(1); pp 59-67
Abstract:
A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo...
The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labeling for NMR spectroscopy
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