Nonnatural amino acids, such as fluorescent amino acids, can be introduced into proteins by using an expanded protein biosynthesis system. This approach requires orthogonal tRNAs that deliver nonnatural amino acids to a ribosome and expanded codon/anticodon pairs, like four-base codon/anticodon pairs. However, in this system, some of nonnatural amino acids are not efficiently incorporated into proteins. To expand the availability of the nonnatural mugatenesis, translation factors and tRNAs must be improved. In this study, we designed and synthesized an elongation factor Tu (EF-Tu) mutant that efficiently binds to tRNAs charged with nonnatural amino acids. We also devised new tRNAs that decode four-base codons and efficiently deliver nonnatural amino acids into ribosome.