Characterization of Protein Domain Function via in vitro DNA Shuffling.

We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the bla CTX-M-14 and bla CTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective approach to identify and characterize important functional domains in bacterial proteins.