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Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction

Abstract Inverse-electron-demand Diels?Alder cycloaddition (DAinv) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of protei...

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Hauptverfasser: Best, Marcel (VerfasserIn) , Degen, Anna (VerfasserIn) , Baalmann, Mathis (VerfasserIn) , Schmidt, Tobias Thomas (VerfasserIn) , Wombacher, Richard (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: May 5, 2015
In: ChemBioChem
Year: 2015, Jahrgang: 16, Heft: 8, Pages: 1158-1162
ISSN:1439-7633
DOI:10.1002/cbic.201500042
Online-Zugang:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1002/cbic.201500042
Verlag, lizenzpflichtig, Volltext: https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cbic.201500042
Volltext
Verfasserangaben:Marcel Best, Anna Degen, Mathis Baalmann, Tobias T. Schmidt, and Richard Wombacher
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Zusammenfassung:Abstract Inverse-electron-demand Diels?Alder cycloaddition (DAinv) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplAW37V), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DAinv?. We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells.
Beschreibung:Gesehen am 22.05.2020
Beschreibung:Online Resource
ISSN:1439-7633
DOI:10.1002/cbic.201500042

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