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Rho GTPases pp 283-303 | Cite as

Use of Phage Display for the Identification of Molecular Sensors Specific for Activated Rho

  • Patrick Chinestra
  • Isabelle Lajoie-Mazenc
  • Jean-Charles Faye
  • Gilles Favre
Part of the Methods in Molecular Biology book series (MIMB, volume 827)

Abstract

We describe a phage display approach to select active Rho-specific scFv sensors. This in vitro technique allows preserving the antigen conformation stability all along the selection process. We used the GTP locked RhoBQ63L mutant as antigen against the Griffin.1 library composed of a human synthetic VH + VL scFv cloned in the pHEN2 phagemid vector. The method described here has permitted to identify an scFv that discriminates between the activated and the inactivated form of the Rho subfamily.

Key words

Phage display scFv Molecular sensors Activated Rho 

Notes

Acknowledgments

We are grateful to Fiona Sait for generously providing the “Griffin.1 Library. We also want to acknowledge Franck Perez for his helpfull advice.

References

  1. 1.
    Etienne-Manneville, S., and Hall, A. (2002) Rho GTPases in cell biology. Nature 420, 629–635.Google Scholar
  2. 2.
    Vega, F.M., and Ridley, A.J. (2008) Rho GTPases in cancer cell biology. FEBS Lett 582, 2093–2101.Google Scholar
  3. 3.
    Aznar, S., Fernandez-Valeron, P., Espina, C., and Lacal, J.C. (2004) Rho GTPases: potential candidates for anticancer therapy. Cancer Lett 206, 181–191.Google Scholar
  4. 4.
    Gomez del Pulgar, T., Benitah, S.A., Valeròn, P.F., Espina, C., and Lacal, J.C. (2005) Rho GTPase expression in tumourigenesis: evidence for a significant link. Bioessays 27, 602–613.Google Scholar
  5. 5.
    Ren, X.D., Kiosses,W.B., and Schwartz, M.A. (1999) Regulation of the small GTP binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18, 578–585.Google Scholar
  6. 6.
    Sander, E.E., van Delft, S., ten Klooster, J.P., Reid, T., van der Kammen, R.A., Michiels, F., and Collard, J.G. (1998) Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol 143, 1385–1398.Google Scholar
  7. 7.
    Goulimari, P., Kitzing, T.M., Knieling, H., Brandt, D.T., Offermanns, S., and Grosse, R. (2005) Gα12/13 is essential for directed cell migration and localized Rho-Dia1 function. J Biol Chem 280, 42242–42251.Google Scholar
  8. 8.
    Bement, W.M., Benink, H.A., and von Dassow, G. (2005) A microtubule-dependent zone of active RhoA during cleavage plane specification. J Cell Biol 170, 91–101.Google Scholar
  9. 9.
    Berger, C.D., Marz, M., Kitzing, T. M., Grosse, R., Steinbeisser, H. (2009) Detection of activated Rho in fixed Xenopus tissue. Dev Dyn 238, 1407–1411.Google Scholar
  10. 10.
    Cascone, I., Audero, E., Giraudo, E., Napione, L., Maniero, F., Philips, M.R., Collard, J.G., Serini, G., and Bussolino, F. (2003) Tie-2-dependent activation of RhoA and Rac1 participates in endothelial cell motility triggered by angiopoietin-1. Blood 102, 2482–2490.Google Scholar
  11. 11.
    Nizak, C., Monier, S., del Nery, E., Moutel, S., Goud, B., and Perez, F. (2003) Recombinant antibodies to the small GTPase Rab6 as conformation sensors. Science 300, 984–987.Google Scholar
  12. 12.
    Horn, I.R., Wittinghofer, A., de Bruïne, A.P., Hoogenboom, H.R. (1999) Selection of phage-displayed fab antibodies on the active conformation of ras yields a high affinity conformation-specific antibody preventing the binding of c-Raf kinase to Ras. FEBS Lett 463, 115–120.Google Scholar
  13. 13.
    Longenecker, K., Read, P., Lin, S.K., Somlyo, A.P., Nakamoto, R.K., Derewenda, Z.S. (2003) Structure of a constitutively activated RhoA mutant (Q63L) at 1.55  Å resolution. Acta Crystallogr D Biol Crystallogr 59, 876–880.Google Scholar
  14. 14.
    Griffiths, A.D., Williams, S.C., Hartley, O., Tomlinson, I.M., Waterhouse, P., Crosby, W.L., Kontermann, R.E., Jones, P.T., Low, N.M., Allison, T.J., Prospero, T.D., Hoogenboom, H.R., Nissim, A., Cox, J.P.L., Harrison, J.L., Zaccolo, M., Gherardi, E., and Winter, G. (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13, 3245–3260.Google Scholar
  15. 15.
    Goffinet, M., Chinestra, P., Lajoie-Mazenc, I., Medale-Giamarchi, C., Favre, G., and Faye, J.C. (2008) Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection. BMC Biotechnol 8, 34–47.Google Scholar
  16. 16.
    Sheffield, P., Garrard, S., and Derewenda, Z. (1999) Overcoming expression and purification problems of RhoGDI using a family of “parallel” expression vectors. Protein Expr Purif 15, 34–39.Google Scholar
  17. 17.
    Dubel, S., Breitling, F., Kontermann, R., Schmidt, T., Skerra, A., and Little, M. (1995) Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J Immunol Methods 178, 201–209.Google Scholar
  18. 18.
    Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and Virnekas, B. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296, 57–86.Google Scholar
  19. 19.
    Silacci, M., Brack, S., Schirru, G., Marlind, J., Ettorre, A., Merlo, A., Viti, F., and Neri, D. (2005) Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5, 2340–2350.Google Scholar
  20. 20.
    Philibert, P., Stoessel, A., Wang, W., Sibler, A.P., Bec, N., Larroque, C., Saven, J.G., Courtete, J., Weiss, E., and Martineau, P. (2007) A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm. BMC Biotechnol 7, 81–97.Google Scholar
  21. 21.
    Tanaka, T., and Rabbitts, T.H. (2003) Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J 22, 1025–1035.Google Scholar
  22. 22.
    Tanaka, T., Williams, R.L., and Rabbitts, T.H. (2007) Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J 26, 3250–3259.Google Scholar
  23. 23.
    Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F., and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem Biophys Res Commun 298, 566–573.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Patrick Chinestra
    • 1
  • Isabelle Lajoie-Mazenc
    • 1
  • Jean-Charles Faye
    • 1
  • Gilles Favre
    • 1
  1. 1.INSERM UMR 1037, Cancer Research Centre of Toulouse, Claudius Regaud Cancer InstituteUniversity of ToulouseToulouseFrance

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