Rapid evolution of a protein in vitro by DNA shuffling | Nature Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement 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) 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 . This is a preview of subscription content, access via your institution Access options Access through your institution Subscription info for Chinese customers We have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal. Go to naturechina.com Buy this article Purchase on SpringerLink Instant access to the full article PDF. 39,95 € Prices may be subject to local taxes which are calculated during checkout Similar content being viewed by others An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo Article Open access 01 July 2025 Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries Article Open access 10 July 2024 Molecular model of TFIIH recruitment to the transcription-coupled repair machinery Article Open access 08 March 2025 References Stemmer, W. P. C. Proc. natn. Acad. Sci. U.S.A. (in the press). Holland, J. H. Scient. Am. 267 , 66–72 (1992). Article Google Scholar Holland, J. H. Adaptation in Natural and Artificial Systems 2nd edn (MIT Press, Cambridge, 1992). Google Scholar Arkin, A. & Youvan, D. C. Proc. natn. Acad. Sci. U.S.A. 89 , 7811–7815 (1992). Article ADS CAS Google Scholar Delagrave, S. & Youvan, D. C. Biotechnology 11 , 1548–1552 (1993). CAS PubMed Google Scholar Palzkill, T. & Botstein, D. J. Bact. 174 , 5237–5243 (1992). Article CAS Google Scholar Oliphant, A. R., Nussbaum, A. L. & Struhl, K. Gene 44 , 177–183 (1986). Article CAS Google Scholar 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 Leung, D. W., Chen, E. & Goeddel, D. V. Technique 1 , 11–15 (1989). Google Scholar Caldwell, R. C. & Joyce, G. F. PCR Meth. Applic. 2 , 28–33 (1992). Article Google Scholar Kauffman, S. A. The Origins of Order (Oxford Univ. Press, New York, 1993). Google Scholar Kauffman, S. A. J. theor. Biol. 157 , 1–7 (1992). Article CAS Google Scholar Bartel, D. P. & Szostak, J. W. Science 261 , 1411–1418 (1993). Article ADS CAS Google Scholar Tuerk, C. & Gold, L. Science 249 , 505–510 (1990). Article ADS CAS Google Scholar Joyce, G. F. Scient. Am. 267 , 90–97 (1992). Article CAS Google Scholar Jacoby, G. A. & Medeiros, A. A. Antimicrob. Ag. Chemother. 35 , 1697–1704 (1991). Article CAS Google Scholar Collatz, E., Labia, R. & Gutmann, L. Molec. Microbiol. 4 , 1615–1620 (1990). Article CAS Google Scholar Philippon, A., Labia, R. & Jacoby, G. Antimicrob. Ag. Chemother. 33 , 1131–1136 (1989). Article CAS Google Scholar McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. Nature 348 , 552–554 (1990). Article ADS CAS Google Scholar Huse, W. D., Sastry, L., Iverson, S. A. & Kang, A. S. Science 246 , 1275–1278 (1989). Article ADS CAS Google Scholar Watson, N. Gene 70 , 399–403 (1988). Article CAS Google Scholar Ambler, R. P. et al. Biochem. J. 276 , 269–272 (1991). Article CAS Google Scholar Download references Author information Authors and Affiliations Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California, 94304, USA Willem P. C. Stemmer Authors Willem P. C. Stemmer View author publications Search author on: PubMed Google Scholar Rights and permissions Reprints and permissions About this article Cite this article Stemmer, W. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370 , 389–391 (1994). https://doi.org/10.1038/370389a0 Download citation Received : 16 February 1994 Accepted : 21 June 1994 Issue date : 04 August 1994 DOI : https://doi.org/10.1038/370389a0 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. 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