Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies

Nature Biotechnology volume 21, pages 1473–1479 (2003)Cite this article

A Retraction to this article was published on 01 May 2004

Abstract

The difficulty of deriving binding ligands to targets identified by genomic sequencing has led to a bottleneck in genomic research. By inserting diverse antibody binding loops into four of the exposed loops at one end of green fluorescent protein (GFP), we have mimicked the natural antibody binding footprint to create robust binding ligands that combine the advantages of antibodies (high affinity and specificity) with those of GFP (intrinsic fluorescence, high stability, expression and solubility). These 'fluorobodies' have been used effectively in enzyme-linked immunosorbent assays (ELISAs), flow cytometry, immuno-fluorescence, arrays and gel shift assays, and show affinities as high as antibodies. Furthermore, the intrinsic fluorescence of fluorobodies correlates with binding activity, allowing the rapid determination of functionality, concentration and affinity. These properties render them especially suitable for the high-throughput genomic scale selections required in proteomics, as well as in diagnostics, target validation and drug development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Fv and GFP footprints.
Figure 2: Tracking functional fluorobodies.
Figure 3: Using fluorobodies.
Figure 4: Using fluorobodies in immunofluorescence.
Figure 5: Fluorobody properties.

Similar content being viewed by others

References

  1. Smith, G.P. Surface presentation of protein epitopes using bacteriophage expression systems. Curr. Opin. Biotechnol. 2, 668–673 (1991).

    Article  CAS  Google Scholar 

  2. Scott, J.K. & Smith, G.P. Searching for peptide ligands with an epitope library. Science 249, 386–390 (1990).

    Article  CAS  Google Scholar 

  3. Sblattero, D. & Bradbury, A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80 (2000).

    Article  CAS  Google Scholar 

  4. Marks, J.D. et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991).

    Article  CAS  Google Scholar 

  5. Vaughan, T.J. et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309–314 (1996).

    Article  CAS  Google Scholar 

  6. Skerra, A. Engineered protein scaffolds for molecular recognition. J. Mol. Recognit. 13, 167–187 (2000).

    Article  CAS  Google Scholar 

  7. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  8. Shi, H. & Wen Su, W. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme Microb. Technol. 28, 25–34 (2001).

    Article  CAS  Google Scholar 

  9. Abedi, M.R., Caponigro, G. & Kamb, A. Green fluorescent protein as a scaffold for intracellular presentation of peptides. Nucleic Acids Res. 26, 623–630 (1998).

    Article  CAS  Google Scholar 

  10. Peelle, B. et al. Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells. Chem. Biol. 8, 521–534 (2001).

    Article  CAS  Google Scholar 

  11. Siegel, M.S. & Isacoff, E.Y. A genetically encoded optical probe of membrane voltage. Neuron 19, 735–741 (1997).

    Article  CAS  Google Scholar 

  12. Doi, N. & Yanagawa, H. Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution. FEBS Lett. 453, 305–307 (1999).

    Article  CAS  Google Scholar 

  13. Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  Google Scholar 

  14. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

    Article  CAS  Google Scholar 

  15. Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985).

    Article  CAS  Google Scholar 

  16. Deane, M. & Norton, J.D. Immunoglobulin gene 'fingerprinting': an approach to analysis of B lymphoid clonality in lymphoproliferative disorders. Br. J. Haematol. 77, 274–281 (1991).

    Article  CAS  Google Scholar 

  17. Lou, J. et al. Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. J. Immunol. Methods 253, 233–242 (2001).

    Article  CAS  Google Scholar 

  18. Scalettar, B.A. et al. Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells. J. Cell Sci. 115, 2399–2412 (2002).

    CAS  PubMed  Google Scholar 

  19. Karlsson, R., Michaelsson, A. & Mattsson, L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J. Immunol. Methods 145, 229–240 (1991).

    Article  CAS  Google Scholar 

  20. Nolan, J.P., Lauer, S., Prossnitz, E.R. & Sklar, L.A. Flow cytometry: a versatile tool for all phases of drug discovery. Drug Discov. Today 4, 173–180 (1999).

    Article  CAS  Google Scholar 

  21. Martineau, P., Jones, P. & Winter, G. Expression of an antibody fragment at high levels in the bacterial cytoplasm. J. Mol. Biol. 280, 117–127 (1998).

    Article  CAS  Google Scholar 

  22. Hink, M.A. et al. Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. J. Biol. Chem. 275, 17556–17560 (2000).

    Article  CAS  Google Scholar 

  23. Reid, B. & Flynn, G. Biochemistry 36, 6786–6791 (1997).

    Article  CAS  Google Scholar 

  24. Griffiths, A.D. et al. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13, 3245–3260 (1994).

    Article  CAS  Google Scholar 

  25. Hufton, S.E. et al. Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands. FEBS Lett. 475, 225–231 (2000).

    Article  CAS  Google Scholar 

  26. van den Beucken, T. et al. Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains. J. Mol. Biol. 310, 591–601 (2001).

    Article  CAS  Google Scholar 

  27. Davies, J. & Riechmann, L. Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. Protein Eng. 9, 531–537 (1996).

    Article  CAS  Google Scholar 

  28. Sblattero, D., Lou, J., Marzari, R. & Bradbury, A. In vivo recombination as a tool to generate molecular diversity in phage antibody libraries. J. Biotechnol. 74, 303–315 (2001).

    CAS  PubMed  Google Scholar 

  29. Sheets, M.D. et al. Efficient construction of a large nonimmune phage antibody library; the production of panels of high affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. USA 95, 6157–6162 (1998).

    Article  CAS  Google Scholar 

  30. Cubbit, A., Woolenweber, L. & Heim, R. Methods Cell Biol. 58, 19–30 (1999).

    Article  Google Scholar 

  31. Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad. Sci. USA 95, 5752–5756 (1998).

    Article  CAS  Google Scholar 

  32. Siegel, R.W., Allen, B., Pavlik, P., Marks, J.D. & Bradbury, A. Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: applications to functional genomics. J. Mol. Biol. 302, 285–293 (2000).

    Article  CAS  Google Scholar 

  33. Visintin, M. et al. The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies. J. Mol. Biol. 317, 73–83 (2002).

    Article  CAS  Google Scholar 

  34. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  35. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  36. Bridge, A.J., Pebernard, S., Ducraux, A., Nicoulaz, A.L. & Iggo, R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34, 263–264 (2003).

    Article  CAS  Google Scholar 

  37. Sledz, C.A., Holko, M., De Veer, M.J., Silverman, R.H. & Williams, B.R. Activation of the interferon system by short-interfering RNAs. Nat. Cell. Biol. 5, 834–839 (2003).

    Article  CAS  Google Scholar 

  38. Nolan, J.P. & Sklar, L.A. The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions. Nat. Biotechnol. 16, 633–638 (1998).

    Article  CAS  Google Scholar 

  39. Lochner, J.E. et al. Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator/green fluorescent protein hybrid. Mol. Biol. Cell 9, 2463–2476 (1998).

    Article  CAS  Google Scholar 

  40. Goslin, K. & Banker, G. in Culturing Nerve Cells (ed. Goslin, G.B.a.K.) 252–281 (MIT Press, Cambridge, MA, USA, 1997).

    Google Scholar 

  41. Scalettar, B.A., Swedlow, J.R., Sedat, J.W. & Agard, D.A. Dispersion, aberration and deconvolution in multi-wavelength fluorescence images. J. Microsc. 182 (Pt 1), 50–60 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to M. Feldhaus, R. Siegel, O. Burrone, R. Sitia, J. Nolan, R. Marzari, D. Sblattero and members of the laboratory for reviewing the manuscript, to N. Velappan, L. Chasteen and P. Dighe for excellent technical assistance and to the National Flow Cytometry Resource (NIH-RR01315) for support and advice. This work was supported by a Genomes to Life, Department of Energy grant DE-FG02-98ER62647 (A.B.), National Institutes of Health grant GM061539 (B.S.) and internal Los Alamos National Laboratory research funds (G.W.).

Author information

Authors and Affiliations

  1. Bioscience Division, HRL-1 TA-43 MS M888, Los Alamos National Laboratory, Los Alamos, 87545, New Mexico, USA

    Ahmet Zeytun, Geoffrey S Waldo & Andrew RM Bradbury

  2. Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    Andreas Jeromin

  3. Department of Physics, Lewis and Clark College, 231 Olin Center, Portland, 97219, Oregon, USA

    Bethe A Scalettar

Authors
  1. Ahmet Zeytun
    View author publications

    Search author on:PubMed Google Scholar

  2. Andreas Jeromin
    View author publications

    Search author on:PubMed Google Scholar

  3. Bethe A Scalettar
    View author publications

    Search author on:PubMed Google Scholar

  4. Geoffrey S Waldo
    View author publications

    Search author on:PubMed Google Scholar

  5. Andrew RM Bradbury
    View author publications

    Search author on:PubMed Google Scholar

Corresponding authors

Correspondence to Geoffrey S Waldo or Andrew RM Bradbury.

Ethics declarations

Competing interests

The technology described in this paper has been patented by A.Z., A.R.M.B. and G.S.W.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zeytun, A., Jeromin, A., Scalettar, B. et al. Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies. Nat Biotechnol 21, 1473–1479 (2003). https://doi.org/10.1038/nbt911

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/nbt911

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing