Site-specific incorporation of unnatural amino acids into proteins in mammalian cells.

Expanding the repertoire of genetically encoded amino acids in cultured mammalian cells requires the expression of the bacterial or archaeal pair of a tRNA and an aminoacyl-tRNA synthetase variant engineered to be specific to the amino acid, along with the supplementation of an unnatural amino acid in the growth medium. The expression of the pair is generally achieved by transfecting the cultured cells with the plasmids bearing the genes encoding the exogenous pair of translation molecules. Here, we provide a description of some of these plasmids and protocols for transfecting cells with the plasmids and preparing growth media supplemented with unnatural amino acids, to facilitate their incorporation into proteins at specific sites.