Abstract The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study of large molecular weight systems). The method consists of pre-induction of the pEVOL plasmid encoding the tRNA/aminoacyl-tRNA synthetase pair in a rich, H2O-based medium prior to exchanging the culture into a D2O-based medium. Our protocol results in high level of isotopic incorporation (~95%) and retains the high expression level of the target protein observed in Luriaâ??Bertani medium.
Content Type Journal Article
Category Communication
Pages 1-5
DOI 10.1007/s10858-012-9606-9
Authors
Vincenzo Venditti, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA
Nicolas L. Fawzi, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA
G. Marius Clore, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA
Amino acid selective unlabeling for sequence specific resonance assignments in proteins
Amino acid selective unlabeling for sequence specific resonance assignments in proteins
Abstract Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative...
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A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
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Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using 13C enrichment and solid state NMR.
Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using 13C enrichment and solid state NMR.
Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using 13C enrichment and solid state NMR.
Comp Biochem Physiol A Mol Integr Physiol. 2011 Jul;159(3):219-24
Authors: Creager MS, Izdebski T, Brooks AE, Lewis RV
Abstract
Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 Ma. Despite precisely balanced mechanical properties,...
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09-02-2011 05:40 PM
Site-specific labeling of proteins with NMR-active unnatural amino acids
Site-specific labeling of proteins with NMR-active unnatural amino acids
Abstract A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling...
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[NMR paper] An AMBER/DYANA/MOLMOL phosphorylated amino acid library set and incorporation into NMR structure calculations.
An AMBER/DYANA/MOLMOL phosphorylated amino acid library set and incorporation into NMR structure calculations.
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J Biomol NMR. 2005 Sep;33(1):15-24
Authors: Craft JW, Legge GB
Protein structure determination using Nuclear Magnetic Resonance (NMR) requires the use of molecular dynamics programs that incorporate both NMR experimental and implicit atomic data. Atomic parameters for each amino acid type are encoded in...
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[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
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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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Automated amino acid side-chain NMR assignment of proteins using 13C- and 15N-resolved 3D [1H,1H]-NOESY
Automated amino acid side-chain NMR assignment of proteins using 13C- and 15N-resolved 3D -NOESY
Francesco Fiorito, Torsten Herrmann, Fred F. Damberger and Kurt Wüthrich
Journal of Biomolecular NMR; 2008; 42(1); pp 23-33
Abstract
ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of 1HN, 15N, 13Cα, 13Cβ and possibly 1Hα from the previous polypeptide backbone assignment, and one or several 3D 13C- or 15N-resolved -NOESY spectra. ASCAN has also been...
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A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
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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...
An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media
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