Precision Labeling of Native Antibodies with Lock Coupling

The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials development, bioimaging, nanomedicine, and other protein-based technologies. Here, we describe Lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate sidechains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at pre-associated amine-acid pairs by creating barriers to diffusive reactivity. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain, and introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and GB1:IgG stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Ab, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys/Asp or Lys/Glu pairs at their interfaces, with potential for precision synthesis of myriad protein-based probes for imaging, biomaterials, biophysics, and medicine.