ArticleJournal of biomolecular NMR2012
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing.
Article in Journal of biomolecular NMR, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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19 citing papers in PubMed.
- Development of SpyTag/SpyCatcher-driven thermostable cyclic laccase and its application in lignin and dye degradation and polymerization of phenolic compounds.Science China. Life sciences · 2026Article
- Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.Protein and peptide letters · 2024Article
- The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.Molecules (Basel, Switzerland) · 2021Article
- Asparaginyl endopeptidases: enzymology, applications and limitations.Organic & biomolecular chemistry · 2021Review
- Conserved allosteric ensembles in disordered proteins using TROSY/anti-TROSY RBiophysical journal · 2021Article
- Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.PloS one · 2021Article
- Chemoenzymatic Semisynthesis of Proteins.Chemical reviews · 2020Review
- NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein.Frontiers in chemistry · 2020Article
- Heavy Enzymes and the Rational Redesign of Protein Catalysts.Chembiochem : a European journal of chemical biology · 2019Article
- Protein Structure Determination in Living Cells.International journal of molecular sciences · 2019Review
- Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation.Journal of biomolecular NMR · 2018Article
- Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.Journal of biomolecular NMR · 2018Article
- Current NMR Techniques for Structure-Based Drug Discovery.Molecules (Basel, Switzerland) · 2018Review
- Dynamic domain arrangement of CheA-CheY complex regulates bacterial thermotaxis, as revealed by NMR.Scientific reports · 2017Article
- Structure-based engineering and comparison of novel split inteins for protein ligation.Molecular bioSystems · 2014Article
- Semi-synthesis of labeled proteins for spectroscopic applications.Molecules (Basel, Switzerland) · 2013Review
- SPLICEFINDER - a fast and easy screening method for active protein trans-splicing positions.PloS one · 2013Article
- Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology.Journal of biomolecular NMR · 2013Article
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5 authors.
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Abstract
Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1-5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation.
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