incorporation of non-natural amino acids into proteins in response to amber or four-base codons is a useful technology for protein research. In the case of the amber codon, however, release factor 1 call competitively decode the same codon, and consequently inhibit the incorporation of non-natural amino acids. To improve amber codon-mediated incorporation, we carried out a comprehensive screening of amber suppressor tRNAs derived from all tRNAs encoded in the genomes of Escherichia call K12 and Mycoplasma capricolum. The amber suppressor tRNAs were synthesized from synthetic genes, aminoacylated with a fluorescent non-natural amino acid, and added to an E. coli cell-free translation system. Fluorescent SDS-PAGE analysis indicated that Trp tRNAs showed high suppressor activity in both organisms. Further mutagenesis and screening revealed that M. capricalum Trp, tRNA with G1C72A73 mutation is the most suitable for efficient and specific incorporation of non-natural amino acids into Proteins in response to the amber codon. (C) 2008 Elsevier Inc. All rights reserved.