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15 N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes

Ozawa, Kiyoshi; Wu, Peter; Dixon, Nicholas; Otting, Gottfried

Description

[15N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [15N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [15N]-HSQC spectra by amino acid type selective [15N]-labelling. The samples can be measured without chromatographic protein purification, dilution of isotopes by...[Show more]

dc.contributor.authorOzawa, Kiyoshi
dc.contributor.authorWu, Peter
dc.contributor.authorDixon, Nicholas
dc.contributor.authorOtting, Gottfried
dc.date.accessioned2015-12-13T23:03:56Z
dc.identifier.issn1742-464X
dc.identifier.urihttp://hdl.handle.net/1885/85147
dc.description.abstract[15N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [15N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [15N]-HSQC spectra by amino acid type selective [15N]-labelling. The samples can be measured without chromatographic protein purification, dilution of isotopes by transaminase activities are suppressed, and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum number of samples for the identification of all [15N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [15N]-HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular, combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h, including protein production and NMR measurement.
dc.publisherBlackwell Publishing Ltd
dc.sourceThe FEBS Journal
dc.subjectKeywords: amino acid; aminotransferase; asparagine; aspartic acid; cell extract; isotope; nitrogen 15; proline; ligand; nitrogen; protein; amino acid analysis; amino acid metabolism; cell free system; cell growth; chromatography; enzyme activity; enzyme repression; 15N-HSQC; 15N-labelled amino acids; Cell-free protein synthesis; Combinatorial labelling; Protein-ligand interactions
dc.title15 N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes
dc.typeJournal article
local.description.notesImported from ARIES
local.description.refereedYes
local.identifier.citationvolume273
dc.date.issued2006
local.identifier.absfor060199 - Biochemistry and Cell Biology not elsewhere classified
local.identifier.absfor100299 - Environmental Biotechnology not elsewhere classified
local.identifier.ariespublicationMigratedxPub13390
local.type.statusPublished Version
local.contributor.affiliationOzawa, Kiyoshi, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationWu, Peter, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationDixon, Nicholas, College of Physical and Mathematical Sciences, ANU
local.contributor.affiliationOtting, Gottfried, College of Physical and Mathematical Sciences, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.issue18
local.bibliographicCitation.startpage4154
local.bibliographicCitation.lastpage4159
local.identifier.doi10.1111/j.1742-4658.2006.05433.x
dc.date.updated2015-12-12T07:52:23Z
local.identifier.scopusID2-s2.0-33748311913
CollectionsANU Research Publications

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