Orthogonal Protein Translation Using Pyrrolysyl-tRNA Synthetases for Single- and Multiple-Noncanonical Amino Acid Mutagenesis.

To date, the two systems most extensively used for noncanonical amino acid (ncAA) incorporation via orthogonal translation are based on the Methanococcus jannaschii TyrRS/tRNA CUA Tyr and the Methanosarcina barkeri/Methanosarcina mazei PylRS/tRNA CUA Pyl pairs. Here, we summarize the development and usage of the pyrrolysine-based system for orthogonal translation, a process that allows for the recombinant production of site-specifically labeled proteins and peptides. Via stop codon suppression in Escherichia coli and mammalian cells, genetically encoded biomolecules can be equipped with a great diversity of chemical functionalities including click chemistry handles, post-translational modifications, and photocaged sidechains.