Nonnatural mutagenesis in E. coli and rabbit reticulocyte lysates by using four-base codons

Various four-base codons had been developed for introduction of nonnatural amino acids into proteins in an E. coli in vitro translation system. Here, the four-base codon mediated incorporation of nonnatural amino acids was investigated in a rabbit reticulocyte lysate. Mutated streptavidin mRNAs containing four-base codons were prepared and expressed in a rabbit reticulocyte lysate in the presence of tRNAs containing corresponding four-base anticodons. Western blot analysis indicates that various four-base codons are efficiently decoded by the corresponding tRNAs in the rabbit in vitro translation system.