Site-specific protein labeling utilizing lipoic acid ligase (LplA) and bioorthogonal inverse electron demand Diels-Alder reaction
Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mut...
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| Main Authors: | , , |
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| Format: | Chapter/Article |
| Language: | English |
| Published: |
06 February 2018
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| In: |
Noncanonical amino acids
Year: 2018, Pages: 365-387 |
| DOI: | 10.1007/978-1-4939-7574-7_23 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1007/978-1-4939-7574-7_23
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| Author Notes: | Mathis Baalmann, Marcel Best, Richard Wombacher |
MARC
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| 520 | |a Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplAW37V) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif—a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DAinv). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness. | ||
| 650 | 4 | |a Bioorthogonal reactions | |
| 650 | 4 | |a Fluorescent probes | |
| 650 | 4 | |a Inverse-electron-demand Diels-Alder cycloaddition | |
| 650 | 4 | |a Ligases | |
| 650 | 4 | |a Site-specific protein labeling | |
| 650 | 4 | |a Tetrazines | |
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| 700 | 1 | |a Wombacher, Richard |e VerfasserIn |0 (DE-588)1025170946 |0 (DE-627)721363288 |0 (DE-576)252266587 |4 aut | |
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