Lipoic Acid Ligase-Promoted Bioorthogonal Protein Modification and Immobilization.

Protein bioconjugation benefits from precise regional and temporal control. One notable way of achieving this control is through the enzymatic attachment of bioorthogonal reactive handles to peptide recognition sequences that are genetically fused to target proteins of interest. The lipoic acid ligase variant, LplA, functionalizes proteins by covalently attaching an azide-bearing lipoic acid derivative to a 13-amino acid recognition sequence known as the lipoic acid ligase acceptor peptide (LAP). Once attached, the azide group can be modified with diverse chemical entities through azide-alkyne click chemistry, enabling conjugation of chemical probes such as fluorophores and facilitating polymer attachment, glycosylation, and protein immobilization in addition to many other possible chemical modifications. The versatility of the attached azide group is complemented by the modular nature of the LAP sequence, which can be introduced within a protein at internal and/or terminal sites as well as at multiple sites simultaneously. In this chapter we describe the in vitro LplA-mediated ligation of 10-azidodecanoic acid to a LAP-containing target protein (i.e., green fluorescent protein (GFP)) and the characterization of the ligation reaction products. Additionally, methods for the modification and immobilization of azide-functionalized LAP-GFP are discussed.