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.

  • Research Article
  • Published:

DNA shuffling of subgenomic sequences of subtilisin

Nature Biotechnology volume 17, pages 893–896 (1999)Cite this article

Abstract

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Phylogenetic subfamilies of 26 subtilisin fragments based on a comparison of amino acid sequences.
Figure 2: Protease activity screens.
Figure 3: Activity distributions of chimeric library.
Figure 4: Multidimensional activity distributions of parental clones and library chimeras.
Figure 5: Parental makeup of the diversified region of selected chimeras based on DNA sequence analysis.

Similar content being viewed by others

References

  1. Darwin, C. On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life (John Murray, London; 1859).

  2. Mann, C.C. Crop scientists seek a new revolution. Science 283, 310–314 (1999).

    Article  CAS  Google Scholar 

  3. Muller, H.J. The relation of recombination to mutational advance. Mutat. Res. 1, 2–9 (1964 ).

    Article  Google Scholar 

  4. Crow, J.F. The odds of losing at genetic roulette. Nature 397, 293–294 (1999).

    Article  CAS  Google Scholar 

  5. Wells, J.A. & Estell, D.A. Subtilisin—an enzyme designed to be engineered. TIBS 13, 291– 297 (1988).

    CAS  PubMed  Google Scholar 

  6. Carter, P., Nilsson, B., Burnier, J.P., Burdick, D. & Wells, J.A. Engineering subtilisin BPN' for site-specific proteolysis. Proteins 6, 240 –248 (1989).

    Article  CAS  Google Scholar 

  7. Pantoliano, M.W. et al. Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding. Biochemistry 28, 7205–7213 ( 1989).

    Article  CAS  Google Scholar 

  8. Bott, R. et al. in Enzyme engineering XI Vol. 672 (eds Clark, D.S. & Estell, D.A.) (The New York Academy of Sciences, New York; 1992).

    Google Scholar 

  9. Graycar, T.P. et al. in Enzyme engineering XI Vol. 672 (eds Clark, D.S. & Estell, D.A.) 71–79 (The New York Academy of Sciences, New York; 1992).

    Google Scholar 

  10. Hastrup, S. et al. Mutated subtilisin genes. PCT Patent Appl. WO 8906279 (Novo Industries, Denmark; 1989).

  11. Mansfeld, J. et al. Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. J. Biol. Chem. 272, 11152 –11156 (1997).

    Article  CAS  Google Scholar 

  12. Bryan, P.N. et al. Proteases of enhanced stability: characterization of a thermostable variant of subtilisin. Proteins 1, 326– 334 (1986).

    Article  CAS  Google Scholar 

  13. Cunningham, B.C. & Wells, J.A. Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure. Protein Eng. 1, 319–325 (1987).

    Article  CAS  Google Scholar 

  14. Kuchner, O. & Arnold, F.H. Directed evolution of enzyme catalysts. Trends Biotechnol. 15, 523– 530 (1997).

    Article  CAS  Google Scholar 

  15. Tange, T., Taguchi, S., Kojima, S., Miura, K. & Momose, H. Improvement of a useful enzyme (subtilisin BPN') by an experimental evolution system. Appl. Microbiol. Biotechnol. 41, 239–244 (1994).

    Article  CAS  Google Scholar 

  16. Kano, H., Taguchi, S. & Momose, H. Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis. Appl. Microbiol. Biotechnol. 47, 46–51 (1997).

    Article  CAS  Google Scholar 

  17. Patkar, S. et al. Effect of mutations in Candida antarctica B lipase. Chem. Phys. Lipids 93, 95–101 (1998).

    Article  CAS  Google Scholar 

  18. Shoichet, B.K., Baase, W.A., Kuroki, R. & Matthews, B.W. A relationship between protein stability and protein function. Proc. Natl. Acad. Sci. USA 92, 452–456 ( 1995).

    Article  CAS  Google Scholar 

  19. Crameri, A., Raillard, S.-A., Bermudez, E. & Stemmer, W.P.C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288– 291 (1998).

    Article  CAS  Google Scholar 

  20. Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 ( 1994).

    Article  CAS  Google Scholar 

  21. Stemmer, W.P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

    Article  CAS  Google Scholar 

  22. Bott, R. & Betzel, C. Subtilisin enzymes (Plenum Press, New York; 1996).

    Book  Google Scholar 

  23. Rao, M.B., Tanksale, A.M., Ghatge, M.S. & Deshpande, V.V. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597– 635 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Siezen, R.J., de Vos, W.M., Leunissen, J.A.M. & Dijkstra, B.W. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteases. Protein Eng. 4 , 719–737 (1991).

    Article  CAS  Google Scholar 

  25. Harwood, C.R. & Cutting, S.M. Molecular biological methods for Bacillus (John Wiley & Sons, Chichester, UK; 1990 ).

    Google Scholar 

  26. Russel, A.J. & Fersht, A.R. Rational modification of enzyme catalysis by engineering surface charge. Nature 328 , 496–500 (1987).

    Article  Google Scholar 

  27. Beebe, A.M., Mauze, S., Schork, N.J. & Coffman, R.L. Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice. Immunity 6, 551– 557 (1997).

    Article  CAS  Google Scholar 

  28. Lamb, C.J., Ryals, J.A., Ward, E.R. & Dixon, R.A. Emerging strategies for enhancing crop resistance to microbial pathogens. Bio/Technology 10, 1436–1445 ( 1992).

    CAS  PubMed  Google Scholar 

  29. Hall, B.G., Yokoyama, S. & Calhoun, D.H. Role of cryptic genes in microbial evolution. Mol. Biol. Evol. 1, 109–124 (1983).

    CAS  PubMed  Google Scholar 

  30. Li, W.H. Retention of cryptic genes in microbial populations. Mol. Biol. Evol. 1, 213–219 ( 1984).

    CAS  PubMed  Google Scholar 

  31. Jones, L.J. et al. Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement. Anal. Biochem. 251, 144–152 (1997).

    Article  CAS  Google Scholar 

  32. Naki, D., Paech, C., Granshaw, G. & Schellenberger, V. Selection of a subtilisin-hyperproducing Bacillus in a highly structured environment. Appl. Microbiol. Biotechnol. 49, 290– 294 (1998).

    Article  CAS  Google Scholar 

  33. Schellenberger, V. Directed evolution can improve subtilisin-secreting Bacillus strains. ASM News 64, 634–638 (1998).

    Google Scholar 

Download references

Acknowledgements

We thank Dr. Titur Kretzschmar for initial amplification of the subtilisin fragments; Andreas Crameri, Steven delCardayre, and Claus Krebber for technical advice; and Sun Ai Raillard for stimulating discussions and comments on the manuscript.

Author information

Author notes

  1. Jon E. Ness and Mark Welch: These authors contributed equally to this work.

Authors and Affiliations

  1. Maxygen, Santa Clara, 95051, 3410 Central Expressway, CA

    Jon E. Ness, Mark Welch & Lori Giver

  2. Enzyme Design, Novo Nordisk, Bagsvaerd, DK 2880, Building 2C , Denmark

    Manuel Bueno & Jeremy Minshull

  3. Novo Nordisk Biotech, Davis, 95616, CA

    Joel R. Cherry & Torben V. Borchert

Authors
  1. Jon E. Ness
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark Welch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lori Giver
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manuel Bueno
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joel R. Cherry
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Torben V. Borchert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Willem P.C. Stemmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeremy Minshull
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeremy Minshull.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ness, J., Welch, M., Giver, L. et al. DNA shuffling of subgenomic sequences of subtilisin. Nat Biotechnol 17, 893–896 (1999). https://doi.org/10.1038/12884

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/12884

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing