Incorporation of noncoded amino acids by in vitro protein biosynthesis

The method of site-specific mutagenesis with noncoded amino acids using sup pression of a nonsense codon by a semi-synthetic tRNA was first introduced in 1989. Initially used to probe the tolerance of the protein biosynthetic machinery for compounds other than the 20 primary amino acids, the method h as since been applied to study a widely diverse range of biological problem s. The ability to introduce side chains bearing subtle structural and elect ronic differences, fluorescent probes, isotope labels, photolabile protecti ng groups, chemical handles and photoactivated cross-linkers at unique site s has facilitated studies not currently accessible by other means. Improvem ents and alternatives to the early methodology are considered as well as so me interesting recent applications.