Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays

  • Mathieu Cinier
  • Marc Petit
  • Frédéric Pecorari
  • Daniel R. Talham
  • Bruno BujoliEmail author
  • Charles TellierEmail author
Original Paper


A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way.


Phosphorylated tag Phosphate cluster Zirconium surface Nanofitin Protein array 



This work was partially supported by grants (no. 04.04.025 and no. 2008.34.0010) from the Délégation Générale à l’Armement (DGA). M.C. was supported by a grant from the DGA. Partial support for this work was also provided by the US National Science Foundation Division of Chemistry under grant no. 0957155 (cofunded by the MPS/CHE and the Office of International Science and Engineering) (DRT). F.P. was supported by a grant from La Région des Pays de la Loire. Mass spectrometry analyses were performed within the BIBS platform located at the INRA Center of Angers-Nantes (INRA, UR1268 Biopolymères Interactions Assemblages, Nantes;


  1. 1.
    Zhou HH, Roy S, Schulman H, Natan MJ (2001) Trends Biotechnol 19:S34–S39PubMedCrossRefGoogle Scholar
  2. 2.
    Feng YF, Ke X, Ma RS, Chen P, Hu GG, Liu FZ (2004) Clin Chem 50:416–422PubMedCrossRefGoogle Scholar
  3. 3.
    Cooper MA (2002) Nat Rev Drug Discov 1:515–528PubMedCrossRefGoogle Scholar
  4. 4.
    Zhu H, Bilgin M, Snyder M (2003) Annu Rev Biochem 72:783–812PubMedCrossRefGoogle Scholar
  5. 5.
    Brody EN, Gold L (2000) J Biotechnol 74:5–13PubMedGoogle Scholar
  6. 6.
    Nygren PA, Skerra A (2004) J Immunol Methods 290:3–28PubMedCrossRefGoogle Scholar
  7. 7.
    Porath J (1988) Trends Anal Chem 7:254–259CrossRefGoogle Scholar
  8. 8.
    Porath J, Carlsson J, Olsson I, Belfrage G (1975) Nature 258:598–599PubMedCrossRefGoogle Scholar
  9. 9.
    Sulkowski E (1985) Trends Biotechnol 3:1–7CrossRefGoogle Scholar
  10. 10.
    Lata S, Reichel A, Brock R, Tampe R, Piehler J (2005) J Am Chem Soc 127:10205–10215PubMedCrossRefGoogle Scholar
  11. 11.
    Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean RA, Gerstein M, Snyder M (2001) Science 293:2101–2105PubMedCrossRefGoogle Scholar
  12. 12.
    Kroger D, Liley M, Schiweck W, Skerra A, Vogel H (1999) Biosens Bioelectron 14:155–161PubMedCrossRefGoogle Scholar
  13. 13.
    Sigal GB, Bamdad C, Barberis A, Strominger J, Whitesides GM (1996) Anal Chem 68:490–497PubMedCrossRefGoogle Scholar
  14. 14.
    Lata S, Piehler J (2005) Anal Chem 77:1096–1105PubMedCrossRefGoogle Scholar
  15. 15.
    Cinier M, Petit M, Williams MN, Fabre RM, Pecorari F, Talham DR, Bujoli B, Tellier C (2009) Bioconjug Chem 20:2270–2277PubMedCrossRefGoogle Scholar
  16. 16.
    Tinazli A, Piehler J, Beuttler M, Guckenberger R, Tampe R (2007) Nat Nanotechnol 2:220–225PubMedCrossRefGoogle Scholar
  17. 17.
    Stensballe A, Andersen S, Jensen ON (2001) Proteomics 1:207–222PubMedCrossRefGoogle Scholar
  18. 18.
    Posewitz MC, Tempst P (1999) Anal Chem 71:2883–2892PubMedCrossRefGoogle Scholar
  19. 19.
    Zhou HJ, Ye ML, Dong J, Han GH, Jiang XN, Wu RN, Zou HF (2008) J Proteome Res 7:3957–3967PubMedCrossRefGoogle Scholar
  20. 20.
    Zhou HJ, Xu SY, Ye ML, Feng S, Pan C, Jiang XG, Li X, Han GH, Fu Y, Zou H (2006) J Proteome Res 5:2431–2437PubMedCrossRefGoogle Scholar
  21. 21.
    Thingholm TE, Jensen ON, Larsen MR (2009) Proteomics 9:1451–1468PubMedCrossRefGoogle Scholar
  22. 22.
    Nonglaton G, Benitez IO, Guisle I, Pipelier M, Leger J, Dubreuil D, Tellier C, Talham DR, Bujoli B (2004) J Am Chem Soc 126:1497–1502PubMedCrossRefGoogle Scholar
  23. 23.
    Lane SM, Monot J, Petit M, Tellier C, Bujoli B, Talham DR (2008) Langmuir 24:7394–7399PubMedCrossRefGoogle Scholar
  24. 24.
    Mouratou B, Schaeffer F, Guilvout I, Tello-Manigne D, Pugsley AP, Alzari PM, Pecorari F (2007) Proc Natl Acad Sci USA 104:17983–17988PubMedCrossRefGoogle Scholar
  25. 25.
    Krehenbrink M, Chami M, Guilvout I, Alzari PM, Pecorari F, Pugsley AP (2008) J Mol Biol 383:1058–1068PubMedCrossRefGoogle Scholar
  26. 26.
    Yamaguchi Y, Wada T, Suzuki F, Takagi T, Hasegawa J, Handa H (1998) Nucleic Acids Res 26:3854–3861PubMedCrossRefGoogle Scholar
  27. 27.
    Maoz R, Sagiv J (1984) J Colloid Interface Sci 100:465–496CrossRefGoogle Scholar
  28. 28.
    Byrd H, Pike JK, Talham DR (1993) Chem Mater 5:709–715CrossRefGoogle Scholar
  29. 29.
    Byrd H, Pike JK, Talham DR (1994) J Am Chem Soc 116:7903–7904CrossRefGoogle Scholar
  30. 30.
    Marin O, Meggio F, Draetta G, Pinna LA (1992) FEBS Lett 301:111–114PubMedCrossRefGoogle Scholar
  31. 31.
    Meggio F, Pinna LA (2003) FASEB J 17:349–368PubMedCrossRefGoogle Scholar
  32. 32.
    Dobrowolska G, Meggio F, Marin O, Lozeman FJ, Li DX, Pinna LA, Krebs EG (1994) FEBS Lett 355:237–241PubMedCrossRefGoogle Scholar
  33. 33.
    Meggio F, Marchiori F, Borin G, Chessa G, Pinna LA (1984) J Biol Chem 259:4576–4579Google Scholar
  34. 34.
    Marin O, Meggio F, Marchiori F, Borin G, Pinna LA (1986) Eur J Biochem 160:239–244PubMedCrossRefGoogle Scholar
  35. 35.
    Monot J, Petit M, Lane SM, Guisle I, Leger J, Tellier C, Talham DR, Bujoli B (2008) J Am Chem Soc 130:6243–6251PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2011

Authors and Affiliations

  • Mathieu Cinier
    • 1
  • Marc Petit
    • 2
  • Frédéric Pecorari
    • 1
  • Daniel R. Talham
    • 3
  • Bruno Bujoli
    • 2
    Email author
  • Charles Tellier
    • 1
    Email author
  1. 1.Laboratoire de Biotechnologie, Biocatalyse et Biorégulation, UFR Sciences et TechniquesUniversité de Nantes, CNRS, UMR 6204Nantes Cedex 3France
  2. 2.Chimie Et Interdisciplinarité: Synthèse Analyse Modélisation (CEISAM)Université de Nantes, CNRS, UMR 6230Nantes Cedex 3France
  3. 3.Department of ChemistryUniversity of FloridaGainesvilleUSA

Personalised recommendations