A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[^(15)N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F,...
A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy
A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy
Abstract
A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-- atoms...
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02-26-2024 09:15 PM
Amino-acid selective isotope labeling enables simultaneous overlapping signal decomposition and information extraction from NMR spectra
Amino-acid selective isotope labeling enables simultaneous overlapping signal decomposition and information extraction from NMR spectra
Abstract
Signal overlapping is a major bottleneck for protein NMR analysis. We propose a new method, stable-isotope-assisted parameter extraction (SiPex), to resolve overlapping signals by a combination of amino-acid selective isotope labeling (AASIL) and tensor decomposition. The basic idea of Sipex is that overlapping signals can be decomposed with the help of intensity patterns derived from quantitative fractional...
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02-29-2020 09:52 PM
An improved protocol for amino acid type-selective isotope labeling in insect cells
An improved protocol for amino acid type-selective isotope labeling in insect cells
Abstract
An improved expression protocol is proposed for amino acid type-specific , -isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449â??456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino...
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07-15-2017 05:05 PM
[NMR paper] NMR conformational dynamics of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.
NMR conformational dynamics of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.
Related Articles NMR conformational dynamics of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.
Biochem Biophys Res Commun. 2014 Jun 2;
Authors: Vourtsis DJ, Chasapis CT, Pairas G, Bentrop D, Spyroulias GA
Abstract
NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent...
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06-20-2014 08:14 PM
[NMR paper] Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary-Button_120x27px_FullText.gif Related Articles Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
Chembiochem. 2013 Jan 30;
Authors: Michel E, Skrisovska L, Wüthrich K, Allain FH
Abstract
Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes...
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02-03-2013 10:19 AM
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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10-05-2011 08:57 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...
A comprehensive assessment of selective amino acid (15)N-labeling in human embryonic kidney 293 cells for NMR spectroscopy
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