Residue-Specific Incorporation of Noncanonical Amino Acids for Protein Engineering

The incorporation of noncanonical amino acids has given protein chemists access to an expanded repertoire of amino acids. This methodology has significantly broadened the scope of protein engineering allowing introduction of amino acids with non-native functionalities, such as bioorthogonal reactive handles (azides and alkynes) and hydrophobic fluorinated side chains. Here, we describe the efficient residue-specific replacement of methionine by azidonorleucine in an engineered green fluorescent protein using a bacterial expression system to introduce a single reactive site for the strain-promoted azide-alkyne cycloaddition.