Site-specific protein and peptide modification by redeploying an asparaginyl ligase for noncanonical reactions

The ability to precisely modify proteins and peptides is fundamental to studying their function and creating new variants or topologies with improved properties. Recent studies have transformed the scope of transpeptidases as versatile tools for site-specific modification of proteins and peptides. The engineered asparaginyl ligase OaAEP1 is an ultrafast transpeptidase that stands out owing to its ability to efficiently catalyze a diverse range of modifications that extend well beyond its natural function to generate backbone cyclic peptides in plants. In this Protocol Extension, we describe a framework for the design and application of noncanonical reactions catalyzed by OaAEP1 that provide access to engineered products with customized terminal or side-chain modifications. The reactions proceed cleanly under mild, nondenaturing conditions and can be applied to a broad array of substrates produced by chemical synthesis or recombinant expression, including folded proteins and peptides. After preparing the required substrates and reagents (~5 d) and expressing the recombinant enzyme in Escherichia coli (~3 d), OaAEP1-catalyzed reactions can be carried out in a matter of minutes to hours. We describe methods for installing non-native C-terminal modifications, including by conjugating commercially available nonpeptidic amines (reactive handles, carbohydrates and so on) or ligating a reversed (retro) substrate mimetic that enables production of genetically inaccessible C-to-C fusions. We also describe procedures for OaAEP1-catalyzed side-chain modification of proteins and peptides, which can be applied to generate side-chain-to-tail macrocyclic products, to label a specific side-chain amine with a dye or other reporter tag, or to produce defined protein-cyclic peptide fusions.