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 mutant of the lipoic acid ligase (LplA ^W37V ) 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 (DA _inv ). 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.