Use of nonsense suppression to generate altered proteins.

The use of suppressed nonsense mutations to generate altered proteins can greatly simplify studies in which a large number of defined mutant proteins are sought. If site-directed mutagenesis is used to generate specific mutations, than for every amber (UAG) mutation constructed, as many as 13 different amino acids can be inserted at the corresponding site in the protein. This allows a rapid screening of many altered proteins for those with interesting properties. Once identified, the interesting substitutions can be regenerated by missense changes, to avoid some of the potential problems of the method. Nonsense suppression has been used to generate more than 3300 amino acid replacements in the E. coli lac repressor, and close to 250 amino acid substitutions in E. coli thymidylate synthase.