p-cyanophenylalanine and 4-(trifluoromethyl)phenylalanine

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

Topics: Alanine; Amino Acyl-tRNA Synthetases; Cell-Free System; Codon, Nonsense; Escherichia coli; Genetic Engineering; Methanococcus; Nitriles; Phenylalanine; Protein Biosynthesis; RNA, Transfer; Suppression, Genetic