Protein evolution by "codon shuffling": A novel method for generating highly variant mutant libraries by assembly of hexamer DNA duplexes

The laboratory evolution of functional proteins holds great fascination as an effective tool for searching and discovering novelty in the protein space. Here, we employ a simple molecular approach for generating a multitude of structurally diverse yet functionally similar proteins that have all "evolved" from the parent enzyme, in this case a beta-lactamase. By randomly combining a set of 14 DNA-hexamer duplexes, each corresponding to judiciously chosen amino acid pairs, we were able to generate functional proteins that contained large regions of previously unknown sequences. Some of the generated proteins were not only more active than the parent, they were also significantly smaller in size. Our approach could therefore be effectively used in searching for novel structural as well as functional proteins.