Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code

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Abstract
One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.