Rapid evolution of a protein in vitro by DNA shuffling (original) (raw)

Nature volume 370, pages 389–391 (1994)Cite this article

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.

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References

  1. Stemmer, W. P. C. Proc. natn. Acad. Sci. U.S.A. (in the press).
  2. Holland, J. H. Scient. Am. 267, 66–72 (1992).
    Article Google Scholar
  3. Holland, J. H. Adaptation in Natural and Artificial Systems 2nd edn (MIT Press, Cambridge, 1992).
    Google Scholar
  4. Arkin, A. & Youvan, D. C. Proc. natn. Acad. Sci. U.S.A. 89, 7811–7815 (1992).
    Article ADS CAS Google Scholar
  5. Delagrave, S. & Youvan, D. C. Biotechnology 11, 1548–1552 (1993).
    CAS PubMed Google Scholar
  6. Palzkill, T. & Botstein, D. J. Bact. 174, 5237–5243 (1992).
    Article CAS Google Scholar
  7. Oliphant, A. R., Nussbaum, A. L. & Struhl, K. Gene 44, 177–183 (1986).
    Article CAS Google Scholar
  8. Hermes, J. D., Blacklow, S. C. & Knowles, J. R. Proc. natn. Acad. Sci. U.S.A. 87, 696–700 (1990).
    Article ADS CAS Google Scholar
  9. Leung, D. W., Chen, E. & Goeddel, D. V. Technique 1, 11–15 (1989).
    Google Scholar
  10. Caldwell, R. C. & Joyce, G. F. PCR Meth. Applic. 2, 28–33 (1992).
    Article Google Scholar
  11. Kauffman, S. A. The Origins of Order (Oxford Univ. Press, New York, 1993).
    Google Scholar
  12. Kauffman, S. A. J. theor. Biol. 157, 1–7 (1992).
    Article CAS Google Scholar
  13. Bartel, D. P. & Szostak, J. W. Science 261, 1411–1418 (1993).
    Article ADS CAS Google Scholar
  14. Tuerk, C. & Gold, L. Science 249, 505–510 (1990).
    Article ADS CAS Google Scholar
  15. Joyce, G. F. Scient. Am. 267, 90–97 (1992).
    Article CAS Google Scholar
  16. Jacoby, G. A. & Medeiros, A. A. Antimicrob. Ag. Chemother. 35, 1697–1704 (1991).
    Article CAS Google Scholar
  17. Collatz, E., Labia, R. & Gutmann, L. Molec. Microbiol. 4, 1615–1620 (1990).
    Article CAS Google Scholar
  18. Philippon, A., Labia, R. & Jacoby, G. Antimicrob. Ag. Chemother. 33, 1131–1136 (1989).
    Article CAS Google Scholar
  19. McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. Nature 348, 552–554 (1990).
    Article ADS CAS Google Scholar
  20. Huse, W. D., Sastry, L., Iverson, S. A. & Kang, A. S. Science 246, 1275–1278 (1989).
    Article ADS CAS Google Scholar
  21. Watson, N. Gene 70, 399–403 (1988).
    Article CAS Google Scholar
  22. Ambler, R. P. et al. Biochem. J. 276, 269–272 (1991).
    Article CAS Google Scholar

Download references

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  1. Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California, 94304, USA
    Willem P. C. Stemmer

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Stemmer, W. Rapid evolution of a protein in vitro by DNA shuffling.Nature 370, 389–391 (1994). https://doi.org/10.1038/370389a0

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