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...
Gespeichert in:
| Hauptverfasser: | , , |
|---|---|
| Dokumenttyp: | Kapitel/Artikel |
| Sprache: | Englisch |
| Veröffentlicht: |
06 February 2018
|
| In: |
Noncanonical amino acids
Year: 2018, Pages: 365-387 |
| DOI: | 10.1007/978-1-4939-7574-7_23 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1007/978-1-4939-7574-7_23
|
| Verfasserangaben: | Mathis Baalmann, Marcel Best, Richard Wombacher |
| Zusammenfassung: | 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. |
|---|---|
| Beschreibung: | Gesehen am 18.06.2020 |
| Beschreibung: | Online Resource |
| ISBN: | 9781493975747 |
| DOI: | 10.1007/978-1-4939-7574-7_23 |