A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code

Several steps have been completed toward the development of a method for th e site specific incorporation of unnatural amino acids into proteins in viv o. Our approach consists of the generation of amber suppressor tRNA/aminoac yl-tRNA synthetase pairs that are orthogonal to all Escherichia coli endoge nous tRNA/synthetase pairs, followed by directed evolution of the orthogona l aminoacyl-tRNA synthetases to alter their amino-acid specificities. A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair in E. coli has b een derived from the Saccharomyces cerevisiae tRNA(Asp) and aspartyl-tRNA s ynthetase, and the in vitro and in vivo characteristics of this pair were d etermined. Two different antibiotic resistance selections were compared usi ng this novel pair in an effort to develop a tunable positive selection for a mutant synthetase capable of charging its cognate suppressor tRNA with a n unnatural amino acid.