Publication Details (including relevant citation information):
Angew. Chem. Int. Ed. 2010, 49(18), 3211-3214
Genetic incorporation of noncanonical amino acids (NAAs) into proteins has flourished. However, the technique in general only allows the incorporation of a single NAA into a single protein because the amber codon is the only one available codon. Herein, we show that the PylRS–pylT pair can be mutated to suppress the ochre UAA codon and the mutated pair can be coupled with an evolved MjTyrRS–MjtRNATyrCUA pair to efficiently incorporate two different NAAs at two defined sites of a single protein inE. coli by both amber and ochre suppressions. This technique will greatly expand the scope of potential applications for the genetic NAA incorporation approach, and it can be applied to install a FRET pair to a protein for conformation and dynamics studies, synthesize proteins with two different post-translational modifications for functional analysis, or generate phage-displayed peptide libraries with the expanded diversity of the displayed peptides.