Strategies forin vitroandin vivotranslation with non-natural amino acids

The use of site-directed mutagenesis (Smith, 1985) to replace amino acids at any chosen position in a protein, coupled with advances in analytical procedures, has greatly advanced our understanding of biological structure-function relationships in recent years. The only limitation of conventional site-directed mutagenesis is that substitutions are restricted to the 20 naturally occurring amino acids. However, the discovery of a 21st amino acid, selenocysteine, and the development of novel in vitro translation techniques have demonstrated that considerably more site-specific replacements are possible during protein engineering. These techniques have already found a wide range of applications and have shown that the translational machinery is able to accommodate an enormously divergent range of aminoacylated tRNAs. Although these techniques are mainly restricted to in vitro systems, recent progress in our understanding of aminoacyl-tRNA synthetase-catalyzed tRNA charging suggests that it may ultimately be possible to extend this technique to growing cells.