Rapid evolution of a protein in vitro by DNA shuffling

Abstract

DNA SHUFFLING is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly¹. Computer simulations called genetic algorithms^2–4have 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 increase⁹.