Development of amber suppressor tRNAs appropriate for incorporation of nonnatural amino acids

Abstract

An amber suppression method has been used for incorporation of nonnatural amino acids into proteins. However, the incorporation efficiency of nonnatural amino acids through an amber codon has been low, which restricts the application of the proteins containing nonnatural amino acids. In this study, we screened a wide variety of amber suppressor tRNAs to discover tRNAs capable to incorporate nonnatural amino acids with high efficiency. To this purpose, synthetic amber suppressor tRNAs of E. coli and Mycoplasma capricolum were screened for the incorporation of a fluorescently labeled nonnatural amino acid in an E. coli cell-free translation system. tRNAs that showed high capability for the incorporation were then mutated not to be aminoacylated by any of endogenous aminoacyl-tRNA synthetases of E. coli and to enhance the incorporation capability. As a result of these investigations, we successfully obtained several amber suppressor tRNAs with high ability for the incorporation of nonnatural amino acids.