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Current approaches to protein site-directed mutagenesis require an independent user operation for each mutation. This can impede large-scale scanning mutagenesis projects such as the mapping of protein interaction surfaces, active sites, or epitopes. It also prevents the creation of protein libraries of defined complexity for directed evolution purposes. Here we present a simple, fast, and effective way to perform scanning codon mutagenesis throughout a protein sequence. The process allows the researcher to define the new codon change, and therefore any amino acid mutation can be achieved. We demonstrate this approach by creating a library of proteins that contain single unnatural amino acid mutations encoded by the amber stop codon, TAG. The mutant proteins generated by this method can be expressed and assayed individually or used together as a mixed population of "rationally diversified" protein sequences.

A General Method for Scanning Unnatural Amino Acid Mutagenesis