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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| Main Authors: | , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
May 5, 2015
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| In: |
ChemBioChem
Year: 2015, Volume: 16, Issue: 8, Pages: 1158-1162 |
| ISSN: | 1439-7633 |
| DOI: | 10.1002/cbic.201500042 |
| Online Access: | 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 
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| Author Notes: | Marcel Best, Anna Degen, Mathis Baalmann, Tobias T. Schmidt, and Richard Wombacher |
| Summary: | 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. |
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| Item Description: | Gesehen am 22.05.2020 |
| Physical Description: | Online Resource |
| ISSN: | 1439-7633 |
| DOI: | 10.1002/cbic.201500042 |