Saturation mutagenesis of proteins, in which an amino acid at a specific site is substituted with each of the other 19 amino acids, is a powerful method for protein analysis and engineering. However, 19 mutated genes have to be prepared to express all possible amino acid-substituted proteins at one site. We previously reported a four-base codon-mediated saturation mutagenesis method for the expression of all 20 amino acid-substituted proteins from one four-base codon-containing gene using 20 types of chemically aminoacylated tRNAs corresponding to the four-base codon. In this study, an improved method for saturation mutagenesis using an amber codon was developed. By combining the use of Escherichia coli-derived amber suppressor tRNAs and chemically aminoacylated Mycoplasma-derived tRNAs, all 20 mutated proteins were successfully expressed from one amber mutant gene in a cell-free translation system. The use of E. coli-derived amber suppressor tRNAs simplified the preparation of the tRNA reagents required for saturation mutagenesis, and also improved the expression of some of the mutated proteins. The expressed mutant proteins were used to evaluate the effect of the amino acid substitutions on the ligand-binding activity. To further expand the possibilities of saturation mutagenesis, a series of nonnatural amino acids analogous to a naturally occurring amino acid was added to the amino acid repertoire. The expanded saturation mutagenesis was utilized to evaluate the effect of a series of atomic-level side chain substitutions on the protein activity. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.