Abstract
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.
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Acknowledgment
This work was supported by funding from the Deutsche Forschungsgemeinschaft DFG (SPP1623, WO 1888/1-2). Furthermore, Mathis Baalmann acknowledges support from the Landesgradiuertenförderung Baden-Württemberg (LGF BW). The authors thank Stefanie Kühn, Hagen Sparka, and Tobias T. Schmidt for technical and experimental support.
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Baalmann, M., Best, M., Wombacher, R. (2018). Site-Specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_23
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DOI: https://doi.org/10.1007/978-1-4939-7574-7_23
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