Incorporation of nonnatural amino acids into proteins through extension of the genetic code

Incorporation of nonnatural amino acids into specific positions of proteins was achieved by using extended codons such as CGGG four-base codon. Aminoacyl-tRNAs having normatural amino acids and four-base anticodons were prepared and added to an E. coli in vitro translation system together with mutated genes containing four-base codons. Product analysis of the in vitro translation demonstrates that four-base codons are successfully decoded by the corresponding aminoacyl-tRNAs and, as a consequence, normatural amino acids are incorporated into desired positions of proteins. By using two independent four-base codons, incorporation of two normatural amino acids into single proteins was achieved for the first time. Five-base codons were also found to be available for the normatural amino acid incorporation. Extension of the amino acid repertoire was investigated by using four-base codons, and a wide variety of nonnatural amino acids were incorporated into proteins. According to the information on amino acid selectivity of ribosomes, novel fluorescently labeled normatural amino acids were designed and utilized for position-specific fluorescence labeling of proteins. The extended codon strategy will allow scientists to construct novel artificial proteins and biomolecular systems.