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:

Improved properties of FLP recombinase evolved by cycling mutagenesis

Nature Biotechnology volume 16, pages 657–662 (1998)Cite this article

Abstract

The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolabilty of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.

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

Similar content being viewed by others

References

  1. Sauer, B. 1994. Site-specific recombination: developments and applications. Curr. Opin. Biotechnol. 5: 521–527.

    Article  CAS  Google Scholar 

  2. Rossant, J. and Nagy, A. 1995. Genome engineering: the new mouse genetics. Nat. Med. 1: 592–594.

    Article  CAS  Google Scholar 

  3. Rajewsky, K., Gu, H., Kühn, R., Betz, U.A., Muller, W., Roes, J., and Schwenk, F. 1996. Conditional gene targeting. J. Clin. Invest. 98: 600–60.

    Article  CAS  Google Scholar 

  4. Kilby, N.J., Snaith, M.R., and Murray, J.A. 1993. Site-specific recombinases: tools for genome engineering. Trends Genet. 9: 413–421.

    Article  CAS  Google Scholar 

  5. Smith, A.J., De Sousa, M.A., Kwabi-Addo, B., Heppell-Parton, A., Impey, H., and Babbitts, P. 1995. A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nat. Genet. 9: 376–38.

    Article  CAS  Google Scholar 

  6. Ramirez Solis, R., Liu, P., and Bradley, A. 1995. Chromosome engineering in mice. Nature 378: 720–724.

    Article  CAS  Google Scholar 

  7. Gu, H., Marth, J.D., Orban, P.C., Mossmann, H., and Rajewsky, K. 1994. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265: 103–106.

    Article  CAS  Google Scholar 

  8. Schwenk, R, Kühn, R., Angrand, P.O., Rajewsky, K., and Stewart, A.F. 1998. Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acids Res. 26: 1427–1432.

    Article  CAS  Google Scholar 

  9. Logie, C., and Stewart, A.F. 1995. Ligand-regulated site-specific recombination. Proc. Natl. Acad. Sci. USA 92: 5940–5944.

    Article  CAS  Google Scholar 

  10. Golic, K.G., and Lindquist, S. 1989. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59: 499–509.

    Article  CAS  Google Scholar 

  11. Gu, H., Zou, Y.R., and Rajewsky, K. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP- mediated gene targeting. Cell 73: 1155–1164.

    Article  CAS  Google Scholar 

  12. Meyers, E.N., Lewandoski, M., and Martin, G.R. 1998. An Fgf8 mutant allelic series generated by Cre-and FLP-mediated recombination. Nat. Gen. 18: 136–141.

    Article  CAS  Google Scholar 

  13. Buchholz, F, Ringrose, L, Angrand, P.O., Rossi, F, and Stewart, A.F. 1996. Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Res. 24: 4256–4262.

    Article  CAS  Google Scholar 

  14. Brookfield, J.F. 1995. Biotechnology: making selection work. Nature 375: 449.

    Article  CAS  Google Scholar 

  15. Moore, J.C., and Arnold, H.A. 1996. Directed evolution of a paranitrobenzyl esterase for aqueous-organic solvents. Nature Biotechnology 14: 458–467.

    Article  CAS  Google Scholar 

  16. Crameri, A., Whitehorn, E.A., Tate, E., and Stemmer, W.P.C. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology 14: 315–319.

    Article  CAS  Google Scholar 

  17. Kuchner, O., and Arnold, F.H. 1997. Directed evolution of enzyme catalysts. Trends in Biotechnology 15: 523–530.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Harford, M.N., and Peeters, M. 1987. Curing of endogenous 2 micron DNA in yeast by recombinant vectors. Curr. Genet. 11: 315–3.

    Article  CAS  Google Scholar 

  20. Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. 1995. Tight regulation modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177: 4121–1130.

    Article  CAS  Google Scholar 

  21. Lebreton, B., Prasad, P.V, Jayaram, M., and Youderian, P. 1988. Mutations that improve the binding of yeast FLP recombinase to its substrate. Genetics 118: 393–400.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Argos, P., Landy, A., Abremski, K., Egan, J.B., Haggard-Ljungquist, E., Hoess, R.H. et al. 1986. The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 5: 433–440.

    Article  CAS  Google Scholar 

  23. Nunes-Duby, S.E., Kwon, H.J., Tirumalai, R.S., Ellenberger, T, and Landy, A. 1998. Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26: 391–406.

    Article  CAS  Google Scholar 

  24. Kwon, H.J., Tirumalai, R., Landy, A., and Ellenberger, T. 1997. Flexibility in DNA recombination: structure of the lambda integrase catalytic core. Science 276: 126–131.

    Article  CAS  Google Scholar 

  25. Hickman, A.B., Waninger, S., Scocca, J.J., and Dyda, F. 1997. Molecular organization in site-specific recombination: the catalytic domain of bacteriophage HP1 integrase at 2.7 Å resolution. Ceff 89: 227–237.

    CAS  Google Scholar 

  26. Quo, R, Gopaul, D.N., and Van Duyne, G.D. 1997. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389: 40.

    Article  Google Scholar 

  27. Subramanya, H.S., Arciszewska, L.K., Baker, R.A., Bird, L.E., Sherratt, D.J., and Wigley, D.B. 1997. Crystal structure of the site-specific recombinase XerD. EMBOJ. 16: 5178–5187.

    Article  CAS  Google Scholar 

  28. Utatsu, I., Sakamoto, S., Imura, T., and Tohe, A. 1987. Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level. J. Bacteriol. 169: 5537–5545.

    Article  CAS  Google Scholar 

  29. Evans, B.R., Chen, J.W., Parsons, R.L., Bauer, T.K., Teplow, D.B., and Jayaram, M. 1990. Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination. J. Biol. Chem. 265: 18504–18510. [published erratum appears in J. Biol. Chem. 266: 7312, 1991.]

    CAS  PubMed  Google Scholar 

  30. Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. 1991. The N-end rule in bacteria. Science 254: 1374–1377.

    Article  CAS  Google Scholar 

  31. Pan, H., Clary, D., and Sadowski, P.O. 1991. Identification of the DNA-binding domain of the FLP recombinase. J. Biol. Chem. 266: 11347–11354.

    CAS  PubMed  Google Scholar 

  32. Vieille, C., and Zeikus, J.G. 1996. Thermozymes: identifying molecular determinants of protein structure and functional stability. Trends in Biotechnology 14: 183–190.

    Article  CAS  Google Scholar 

  33. Watanabe, K., Chishiro, K., Kitamura, K., and Suzuki, Y. 1991. Praline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosi-dasius KP1006. J. Biol. Chem. 266: 24287–24294.

    CAS  PubMed  Google Scholar 

  34. Burdette, D.S., Vieille, C., and Zeikus, J.G. 1996. Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme. Biochem. J. 316: 115–122.

    Article  CAS  Google Scholar 

  35. Meyer-Leon, L., Gates, C.A., Attwood, J.M., Wood, E.A., and Cox, M.M. 1987. Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system. Nucleic Acids Res. 15: 6469–6488.

    Article  CAS  Google Scholar 

  36. Marshall, E. 1997. The mouse that prompted a roar. Science 277: 24–25.

    Article  CAS  Google Scholar 

  37. O'Gorman, S., and Wahl, G.M. 1997. Mouse engineering. Science 277: 1116–1117.

    Article  Google Scholar 

  38. Heidmann, S., Seifert, W., Kessler, C., and Domdey, H. 1989. Cloning characterization and heterologous expression of the Smal restriction-modification system. Nucleic Acids Res. 17: 9783–9796.

    Article  CAS  Google Scholar 

  39. Buchholz, R, Angrand, P. -O., and Stewart, A.F. 1996. A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs. Nucleic Acids Res. 24: 3118–3119.

    Article  CAS  Google Scholar 

  40. Fromant, M., Blanquet, S., and Plateau, P. 1995. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal. Biochem. 224: 347–353.

    Article  CAS  Google Scholar 

  41. Vartanian, J.P., Henry, M., and Wain Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res. 24: 2627–2631.

    Article  CAS  Google Scholar 

  42. Lorimer, I.A., and Pastan, I. 1995. Random recombination of antibody single chain Fv sequences after fragmentation with DNasel in the presence of Mn2+. Nucleic Acids Res. 23: 3067–3068.

    Article  CAS  Google Scholar 

  43. Wierzbicki, A., Kendall, M., Abremski, K., and Hoess, R. 1987. A mutational analysis of the bacteriophage P1 recombinase Cre. J. Mol. Biol. 195: 785–794.

    Article  CAS  Google Scholar 

  44. Nagy, A., Rossant, J., Nagy, R., Abramow Newerly, W., and Roder, J.C. 1993. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90: 8424–8428.

    Article  CAS  Google Scholar 

  45. Kellendonk, C., Tranche, R, Monaghan, A.P., Angrand, P.O., Stweart, A.R, and Schütz, G. 1996. Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucleic Acids Res. 24: 1404–1411.

    Article  CAS  Google Scholar 

  46. Rost, B., Sander, C., and Schneider, R. 1994. PHD—an automatic mail server for protein secondary structure prediction. Comput. Appl. Biosci. 10: 53–60.

    CAS  Google Scholar 

Download references

Author information

Author notes

  1. A. Francis Stewart: (e-mail: stewart@embl-heidelberg.de)

Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Gene Expression Program, Meyerhofstrasse 1, D-69117, Heidelberg, Germany

    Frank Buchholz, Pierre-Olivier Angrand & A. Francis Stewart

Authors
  1. Frank Buchholz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pierre-Olivier Angrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Francis Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Buchholz, F., Angrand, PO. & Stewart, A. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16, 657–662 (1998). https://doi.org/10.1038/nbt0798-657

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0798-657

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