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