RAPID EVOLUTION OF A PROTEIN IN-VITRO BY DNA SHUFFLING

DNA shuffling is a method for in vitro homologous recombination of poo ls of selected mutant genes by random fragmentation and polymerase cha in reaction (PCR) reassembly(1). Computer simulations called genetic a lgorithms(2-4) have demonstrated the importance of iterative homologou s recombination for sequence evolution. Oligonucleotide cassette mutag enesis(5-11) and error-prone PCR are not combinatorial and thus are li mited in searching sequence space(1,14). We have tested mutagenic DNA shuffling for molecular evolution(14-18) in a beta-lactamase model sys tem(9,19). Three cycles of shuffling and two cycles of backcrossing wi th wild-type DNA, to eliminate non-essential mutations, were each foll owed by selection on increasing concentrations of the antibiotic cefot axime. We report here that selected mutants had a minimum inhibitory c oncentration of 640 mu g ml(-1), a 32,000-fold increase and 64-fold gr eater than any published TEM-1 derived enzyme. Cassette mutagenesis an d error-prone PCR resulted in only a 16-fold increase(9).