Molecular Biotechnology

, Volume 46, Issue 1, pp 58–62

Characterisation of a DNA Polymerase Highly Mutated Along the Template Binding Interface



Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant’s broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.


Reverse transcriptase Enzyme engineering In vitro selection Phage display Directed evolution 


  1. 1.
    Smith, G. P. (1985). Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science, 228, 1315–1317.CrossRefGoogle Scholar
  2. 2.
    Marks, J. D., Hoogenboom, H. R., Griffiths, A. D., & Winter, G. (1992). Molecular evolution of proteins on filamentous phage. Mimicking the strategy of the immune system. Journal of Biological Chemistry, 267, 16007–16010.Google Scholar
  3. 3.
    Jestin, J. L. (2008). Functional cloning by phage display. Biochimie, 90, 1273–1278.CrossRefGoogle Scholar
  4. 4.
    Kristensen, P., & Winter, G. (1998). Proteolytic selection for protein folding using filamentous bacteriophages. Folding & Design, 3, 321–328.CrossRefGoogle Scholar
  5. 5.
    Sieber, V., Pluckthun, A., & Schmid, F. X. (1998). Selecting proteins with improved stability by a phage-based method. Nature Biotechnology, 16, 955–960.CrossRefGoogle Scholar
  6. 6.
    Fastrez, J. (2002). Investigation of phage display for the directed evolution of enzymes. In S. Brakmann and K. Johnsson (Ed.), Directed molecular evolution of proteins (pp. 79–110). Weinheim: Wiley VCH.Google Scholar
  7. 7.
    Jestin, J. L., & Kaminski, P. A. (2004). Directed enzyme evolution and selections for catalysis based on product formation. Journal of Biotechnology, 113, 85–103.CrossRefGoogle Scholar
  8. 8.
    Pedersen, H., Hölder, S., Sutherlin, D. P., Schwitter, U., King, D. S., & Schultz, P. G. (1998). A method for directed evolution and functional cloning of enzymes. Proceedings of the National Academy of Sciences of the USA, 95, 10523–10528.CrossRefGoogle Scholar
  9. 9.
    Jestin, J. L., Kristensen, P., & Winter, G. (1999). A method for the selection of catalytic activity using phage display and proximity coupling. Angewandte Chemie International Edition, 38, 1124–1127.CrossRefGoogle Scholar
  10. 10.
    Demartis, S., Huber, A., Viti, F., Lozzi, L., Giovannoni, L., Neri, P., et al. (1999). A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage. Journal of Molecular Biology, 286, 617–633.CrossRefGoogle Scholar
  11. 11.
    Ponsard, I., Galleni, M., Soumillion, P., & Fastrez, J. (2001). Selection of metalloenzymes by catalytic activity using phage display and catalytic elution. ChemBioChem, 2, 253–259.CrossRefGoogle Scholar
  12. 12.
    Atwell, S., & Wells, J. A. (1999). Selection for improved subtiligases by phage display. Proceedings of the National Academy of Sciences of the USA, 96, 9497–9502.CrossRefGoogle Scholar
  13. 13.
    Heinis, C., Huber, A., Demartis, S., Bertschinger, J., Melkko, S., Lozzi, L., et al. (2001). Selection of catalytically active biotin ligase and trypsin mutants by phage display. Protein Engineering, 14, 1043–1052.CrossRefGoogle Scholar
  14. 14.
    Xia, G., Chen, L., Sera, T., Fa, M., Schultz, P. G., & Romesberg, F. E. (2002). Directed evolution of novel polymerase activities: mutation of a DNA polymerase into an efficient RNA polymerase. Proceedings of the National Academy of Sciences of the USA, 99, 6597–6602.CrossRefGoogle Scholar
  15. 15.
    Fa, M., Radeghieri, A., Henry, A. A., & Romesberg, F. E. (2004). Expanding the substrate repertoire of a DNA polymerase by directed evolution. Journal of the American Chemical Society, 126, 1748–1754.CrossRefGoogle Scholar
  16. 16.
    Jestin, J. L., and Vichier-Guerre, S. (2008). Methods for obtaining thermostable enzymes. Patent US2008014609.Google Scholar
  17. 17.
    Vichier-Guerre, S., Ferris, S., Auberger, N., Mahiddine, K., & Jestin, J. L. (2006). A population of thermostable reverse transcriptases evolved from Thermus aquaticus DNA polymerase I by phage display. Angewandte Chemie International Edition, 45, 6133–6137.CrossRefGoogle Scholar
  18. 18.
    Orsi, E., & Jestin, J. L. (2003). Optimisation of in vitro enzyme selection. Comptes Rendus Chimie, 6, 501–506.CrossRefGoogle Scholar
  19. 19.
    Vichier-Guerre, S., & Jestin, J. L. (2003). Iterative cycles of in vitro protein selection for DNA polymerase activity. Biocatalysis & Biotransformations, 21, 75–78.Google Scholar
  20. 20.
    Jestin, J. L., & Vichier-Guerre, S. (2005). How to broaden enzyme substrate specificity: Strategies, implications and applications. Research in Microbiology, 156, 961–966.CrossRefGoogle Scholar
  21. 21.
    Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J., Hudson, P., & Winter, G. (1991). Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody Fab heavy and light chains. Nucleic Acids Research, 19, 4133–4137.CrossRefGoogle Scholar
  22. 22.
    Jestin, J. L., Volioti, G., & Winter, G. (2001). Improving the display of proteins on filamentous phage. Research in Microbiology, 152, 187–191.CrossRefGoogle Scholar
  23. 23.
    Strobel, H., Ladant, D., & Jestin, J. L. (2003). Efficient display of two enzymes on filamentous phage using an improved signal sequence. Molecular Biotechnology, 24, 1–9.CrossRefGoogle Scholar
  24. 24.
    Bahrami, F., & Jestin, J. L. (2008). Streptococcus agalactiae DNA polymerase I is an efficient reverse transcriptase. Biochimie, 90, 1796–1799.CrossRefGoogle Scholar
  25. 25.
    Pace, N. C., Vajdos, F., Fee, L., Grimsley, G., & Grey, T. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Science, 4, 2411–2423.CrossRefGoogle Scholar
  26. 26.
    Doublié, S., Tabor, S., Long, A. M., Richardson, C. C., & Ellenberger, T. (1998). Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 angström resolution. Nature, 391, 251–258.CrossRefGoogle Scholar
  27. 27.
    Patel, P. H., & Loeb, L. A. (2000). DNA polymerase active site is highly mutable: Evolutionary consequences. Proceedings of the National Academy of Sciences of the USA, 97, 5095–5100.CrossRefGoogle Scholar
  28. 28.
    Delarue, M., Poch, O., Tordo, N., Moras, D., & Argos, P. (1990). An attempt to unify the structure of polymerases. Protein Engineering, 6, 461–467.Google Scholar
  29. 29.
    Ghadessy, F. J., Ong, J. L., & Holliger, P. (2001). Directed evolution of polymerase function by compartmentalised self-replication. Proceedings of the National Academy of Sciences of the USA, 98, 4552–4557.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Département de Biologie Structurale et ChimieInstitut PasteurParis 15France

Personalised recommendations