Site-specific incorporation of two noncanonical amino acids for two-color bioorthogonal labeling and chemical-controlled crosslinking of proteins on live mammalian cells

The pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) is widely used in protein engineering to site-specifically introduce noncanonical amino acids (ncAAs) through nonsense codon suppression. Here, we engineer the PylT/RS pair encoded by Methanogenic archaeon ISO4-G1 (G1) to be orthogonal to Mma PylT/RS and alter the PylRS active site to accept a complementary ncAA spectrum. We combine the resulting mutual orthogonal pairs for site-specific dual ncAA incorporation at unprecedented efficiency. Demonstrating the robustness of the system, we incorporate two ncAAs with compatible bioorthogonal reactivity into a Notch receptor, as well as a G-protein coupled receptor. We show that selective and site-specific incorporation of two ncAAs allows for two-color bioorthogonal labeling as well as chemical-controlled crosslinking of surface proteins on live mammalian cells. This dataset contains plasmid sequences, additional figures and primary data related to the associated publication.