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An activity-independent selection system of thermostable protein variants

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Abstract

We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

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Figure 1: Kattv-fused IFNγ variants induce T. thermophilus growth relative to their thermostability level.
Figure 2: Thermoresistance kinetics of two thermostable mutants of IFNα, IFNβ and IFNγ.

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References

  1. Eijsink, V.G., Gaseidnes, S., Borchert, T.V. & van den Burg, B. Biomol. Eng. 22, 21–30 (2005).

    Article  CAS  Google Scholar 

  2. Shusta, E.V., Kieke, M.C., Parke, E., Kranz, D.M. & Wittrup, K.D. J. Mol. Biol. 292, 949–956 (1999).

    Article  CAS  Google Scholar 

  3. Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M. & Wittrup, K.D. Nat. Biotechnol. 18, 754–759 (2000).

    Article  CAS  Google Scholar 

  4. Bommarius, A.S., Broering, J.M., Chaparro-Riggers, J.F. & Polizzi, K.M. Curr. Opin. Biotechnol. 17, 606–610 (2006).

    Article  CAS  Google Scholar 

  5. Matsumura, M. & Aiba, S. J. Biol. Chem. 260, 15298–15303 (1985).

    CAS  PubMed  Google Scholar 

  6. Waldo, G.S., Standish, B.M., Berendzen, J. & Terwilliger, T.C. Nat. Biotechnol. 17, 691–695 (1999).

    Article  CAS  Google Scholar 

  7. Maxwell, K.L., Mittermaier, A.K., Forman-Kay, J.D. & Davidson, A.R. Protein Sci. 8, 1908–1911 (1999).

    Article  CAS  Google Scholar 

  8. Slodowski, O., Bohm, J., Schone, B. & Otto, B. Eur. J. Biochem. 202, 1133–1140 (1991).

    Article  CAS  Google Scholar 

  9. Waschutza, G. et al. Protein Eng. 9, 905–912 (1996).

    Article  CAS  Google Scholar 

  10. Saboulard, D. et al. Biotechniques 39, 363–368 (2005).

    Article  CAS  Google Scholar 

  11. Delcourt, M. & Blesa, S. Method for massive mutagenesis. European patent 1311670 B1 and US patent 7202086 B2 (2001).

  12. Cabantous, S. et al. J. Struct. Funct. Genomics 6, 113–119 (2005).

    Article  CAS  Google Scholar 

  13. Park, C. & Marqusee, S. Nat. Methods 2, 207–212 (2005).

    Article  CAS  Google Scholar 

  14. Beeby, M. et al. PLoS Biol. 3, e309 (2005).

    Article  Google Scholar 

  15. Sieber, V., Pluckthun, A. & Schmid, F.X. Nat. Biotechnol. 16, 955–960 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Blésa, J. Sylvestre, B. Winter and J.-C. Barale for useful discussions and comments on the manuscript and M.-A. Carriço, E. Matthieu and S. Jovelin for their technical help. We acknowledge financial support of projects BIO2004-02671 from Ministerio de Educación y Ciencia (MEC), S0505/PPQ/0344 from Comunidad Autónoma de Madrid to José Berenguer and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. E.B.-G. was supported by a fellowship from the MEC.

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Authors and Affiliations

  1. Biométhodes SA, Genavenir 8, 5 rue Henri Desbruères, Evry, 91030, France

    Hélène Chautard, Thierry Menguy, Laure Grand'Moursel & Marc Delcourt

  2. Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, 28049, Spain

    Emilio Blas-Galindo, Felipe Cava & José Berenguer

Authors
  1. Hélène Chautard
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  2. Emilio Blas-Galindo
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  3. Thierry Menguy
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  4. Laure Grand'Moursel
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  5. Felipe Cava
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  6. José Berenguer
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  7. Marc Delcourt
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Contributions

H.C. contributed to design of libraries and construction and selection procedures. E.B.-G. and F.C. contributed to pNCK construction and initial settings and validation of the selection procedures in T. thermophilus. T.M. and L.G.'M. contributed to interferon and enzyme assays. M.D. and J.B. contributed to THR design and determining experimental settings.

Corresponding authors

Correspondence to José Berenguer or Marc Delcourt.

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Competing interests

H.C., T.M. L.G.'M. and M.D. are employed by a private company, Biomethodes SA.

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Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–2, Supplementary Methods (PDF 254 kb)

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Chautard, H., Blas-Galindo, E., Menguy, T. et al. An activity-independent selection system of thermostable protein variants. Nat Methods 4, 919–921 (2007). https://doi.org/10.1038/nmeth1090

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  • DOI: https://doi.org/10.1038/nmeth1090

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