Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids

AbstractIt has previously been demonstrated that the unnatural amino acid p-Cl-phenylalanine can be attached to tRNAPhe by a modified phenylalanyl-tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p-Cl- or p-Br-phenylalanine into full-length luciferase in vitro. The incorporation of p-Cl-phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery