Creating site-specific isopeptide linkages between proteins with the traceless Staudinger ligation

Site-specific isopeptide linkages between the ε-amino group of a lysine residue in one protein and a carboxyl group in another are central to ubiquitin-mediated protein degradation and other cellular processes. These linkages are inaccessible with common recombinant DNA techniques. Here, we describe a method to link two proteins by an authentic isopeptide bond. The method unites three techniques at the forefront of molecular biology. An azidonorleucine residue is installed at a desired site in a substrate protein by nonnatural amino acid incorporation, and a phosphinothioester is installed at the C terminus of a pendant protein by expressed protein ligation. Then, the traceless Staudinger ligation is used to link the substrate and pendant proteins via an isopeptide bond. This method facilitates the study of otherwise intractable protein structure-function relationships.