Rapid evolution of a protein in vitro by DNA shuffling

DNA SHUFFLING is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly 1 . Computer simulations called genetic algorithms 2–4 have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis 5–11 and error-prone PCR 12,13 are not combinatorial and thus are limited in searching sequence space 1,14 . We have tested mutagenic DNA shuffling for molecular evolution 14–18 in a p-lactamase model system 9,19 . Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 μg ml -1 , a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase 9 .