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 algorithms2-4have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis5-11 and error-prone PCR12,13 are not combinatorial and thus are limited in searching sequence space1,14. We have tested mutagenic DNA shuffling for molecular evolution14-18 in a p-lactamase model system9,19. 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.
- Publication:
-
Nature
- Pub Date:
- August 1994
- DOI:
- 10.1038/370389a0
- Bibcode:
- 1994Natur.370..389S