Molecular Biotechnology

, Volume 54, Issue 3, pp 829–841 | Cite as

Directed Evolution of a Fluorogen-Activating Single Chain Antibody for Function and Enhanced Brightness in the Cytoplasm

  • Bradley P. Yates
  • Michelle A. Peck
  • Peter B. Berget


Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP–fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.


Directed evolution Fluorogen activating protein Fluoromodule Intrabody scFv Fluorogenic dyes 


  1. 1.
    Szent-Gyorgyi, C., et al. (2008). Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nature Biotechnology, 26, 235–240.CrossRefGoogle Scholar
  2. 2.
    Feldhaus, M. J., et al. (2003). Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nature Biotechnology, 21, 163–170.CrossRefGoogle Scholar
  3. 3.
    Ozhalici-Unal, H., et al. (2008). A rainbow of fluoromodules: A promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes. Journal of the American Chemical Society, 130, 12620–12621.CrossRefGoogle Scholar
  4. 4.
    Silva, G., et al. (2007). Experimental and computational investigation of unsymmetrical cyanine dyes: understanding torsionally responsive fluorogenic dyes. Journal of the American Chemical Society, 129, 5710–5718.CrossRefGoogle Scholar
  5. 5.
    Fitzpatrick, J., et al. (2009). STED nanoscopy in living cells using fluorogen activating proteins. Bioconjugate Chemistry, 20, 1843–1847.CrossRefGoogle Scholar
  6. 6.
    Fisher, G. W., et al. (2010). Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology. Journal of Biomolecular Screening, 15, 703–709.CrossRefGoogle Scholar
  7. 7.
    Glover, M., et al. (2012). Pharmacological rescue of mutant CFTR detected using a novel fluorescence platform. Molecular Medicine, 18, 685–696.Google Scholar
  8. 8.
    Holleran, J., et al. (2010). Fluorogen-activating proteins as biosensors of cell surface proteins in living cells. Cytometry Part A, 77, 776–782.CrossRefGoogle Scholar
  9. 9.
    Glockshuber, R., Schmidt, T., & Pluckthun, A. (1992). The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli. Biochemistry, 31, 1270–1279.CrossRefGoogle Scholar
  10. 10.
    Proba, K., Honegger, A., & Plückthun, A. (1997). A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding. Journal of Molecular Biology, 265, 161–172.CrossRefGoogle Scholar
  11. 11.
    Biocca, S., et al. (1995). Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria. Biotechnology (N.Y.), 13, 1110–1115.CrossRefGoogle Scholar
  12. 12.
    Zhou, C., & Przedborski, S. (2009). Intrabody and Parkinson’s disease. BBA, 1792(7), 634–642.CrossRefGoogle Scholar
  13. 13.
    Dunn, S., et al. (2006). Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide MHC without increasing apparent. Protein Science, 15, 710–721.CrossRefGoogle Scholar
  14. 14.
    Persson, H., et al. (2008). In vitro evolution of an antibody fragment population to find high-affinity hapten binders. Protein Engineering, Design & Selection, 21, 485–493.CrossRefGoogle Scholar
  15. 15.
    Hanes, J., & Plückthun, A. (1997). In vitro selection and evolution of functional proteins by using ribosome display. Proceedings of the National Academy of Sciences of the United States of America, 94, 4937–4942.CrossRefGoogle Scholar
  16. 16.
    Zahnd, C., et al. (2004). Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. The Journal of Biological Chemistry, 279, 18870–18877.CrossRefGoogle Scholar
  17. 17.
    Richman, S., Kranz, D., & Stone, J. (2009). Biosensor detection systems: Engineering stable, high-affinity bioreceptors by yeast surface display. Methods in Molecular Biology, 504, 323–350.CrossRefGoogle Scholar
  18. 18.
    Colby, D. W., et al. (2004). Development of a human light chain variable domain (VL) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display. Journal of Molecular Biology, 342, 901–912.CrossRefGoogle Scholar
  19. 19.
    Alper, H., et al. (2005). Tuning genetic control through promoter engineering. Proceedings of the National Academy of Sciences of the United States of America, 102, 12678–12683.CrossRefGoogle Scholar
  20. 20.
    Song, L., Siguier, B., Dumon, C., Bozonnet, S., & O’Donohue, M. J. (2012). Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy. Biotechnology for Biofuels, 5, 3–18.CrossRefGoogle Scholar
  21. 21.
    Colby, D. W., et al. (2004). Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proceedings of the National Academy of Sciences of the United States of America, 101, 17616–17621.CrossRefGoogle Scholar
  22. 22.
    Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. A., & Foeller, C. (1991). Sequences of proteins of immunological interest. Bethesda, MD: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health.Google Scholar
  23. 23.
    Chao, G., et al. (2006). Isolating and engineering human antibodies using yeast surface display. Nature Protocols, 1, 755–768.CrossRefGoogle Scholar
  24. 24.
    Orr-Weaver, T. L., & Szostak, J. W. (1983). Yeast recombination: The association between double-strand gap repair and crossing-over. Proceedings of the National Academy of Sciences of the United States of America, 80, 4417–4421.CrossRefGoogle Scholar
  25. 25.
    Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9, 671–675.CrossRefGoogle Scholar
  26. 26.
    Visintin, M., et al. (1999). Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proceedings of the National Academy of Sciences of the United States of America, 96, 11723–11728.CrossRefGoogle Scholar
  27. 27.
    Wörn, A., & Plückthun, A. (2001). Stability engineering of antibody single-chain Fv fragments. Journal of Molecular Biology, 305, 989–1010.CrossRefGoogle Scholar
  28. 28.
    Wörn, A., & Plückthun, A. (1999). Different equilibrium stability behavior of ScFv fragments: Identification, classification, and improvement by protein engineering. Biochemistry, 38, 8739–8750.CrossRefGoogle Scholar
  29. 29.
    Wang, T., & Duan, Y. (2011). Probing the stability-limiting regions of an antibody single-chain variable fragment: A molecular dynamics simulation study. Protein Engineering, Design & Selection, 24, 649–657.CrossRefGoogle Scholar
  30. 30.
    MacCallum, R. M., Martin, A. C., & Thornton, J. M. (1996). Antibody-antigen interactions: Contact analysis and binding site topography. Journal of Molecular Biology, 262, 732–745.CrossRefGoogle Scholar
  31. 31.
    Zhu, X., et al. (2004). The origin of enantioselectivity in aldolase antibodies: Crystal structure, site-directed mutagenesis, and computational analysis. Journal of Molecular Biology, 343, 1269–1280.CrossRefGoogle Scholar
  32. 32.
    Golinelli-Pimpaneau, B. (2000). Novel reactions catalysed by antibodies. Current Opinion in Structural Biology, 10, 697–708.CrossRefGoogle Scholar
  33. 33.
    Martin, A. C. R. (2012). Abysis amino acid distributions. Division of Biosciences, University College London. Retrieved August 27, 2012 from
  34. 34.
    Xiang, J., et al. (1999). Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Protein Engineering, 12, 417–421.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Bradley P. Yates
    • 1
  • Michelle A. Peck
    • 1
  • Peter B. Berget
    • 1
    • 2
  1. 1.Department of Biological Sciences & Molecular Biosensor and Imaging CenterCarnegie Mellon UniversityPittsburghUSA
  2. 2.Department of Biological SciencesThe University of the SciencesPhiladelphiaUSA

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