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Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro

Nature Protocols volume 1, pages 714–719 (2006)Cite this article

Abstract

Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment is cloned and overexpressed in E. coli as a fusion with self-splitting intein. After solubilizing and refolding these fusions from inclusion bodies, both EGFP fragments are cleaved from intein and purified using chitin columns. When these EGFP fragments are linked with the two complementary oligonucleotides and combined in equimolar amounts, fluorescence develops within a few minutes. The isolation of profluorescent protein fragments from recombinant E. coli cells requires ∼3 d, and their conjugation to oligonucleotides requires 1–4 h.

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Figure 1: The principle of protein fluorescence complementation.
Figure 2: Experimental design used here for cloning and isolating the two fragments of split EGFP.
Figure 3
Figure 4: Fluorescent response kinetics of the split EGFP system upon DNA hybridization recorded at 524 nm.

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References

  1. Ghosh, I., Hamilton, A.D. & Regan, L. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000).

    Article  CAS  Google Scholar 

  2. Stains, C.I., Porter, J.R., Ooi, A.T., Segal, D.J. & Ghosh, I. DNA sequence-enabled reassembly of the green fluorescent protein. J. Am. Chem. Soc. 127, 10782–10783 (2005).

    Article  CAS  Google Scholar 

  3. Jeong, J. et al. Monitoring of conformational change in maltose binding protein using split green fluorescent protein. Biochem. Biophys. Res. Commun. 339, 647–651 (2006).

    Article  CAS  Google Scholar 

  4. Shyu, Y.J., Liu, H., Deng, X. & Hu, C.D. Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. BioTechniques 40, 61–66 (2006).

    Article  CAS  Google Scholar 

  5. Reid, B.G. & Flynn, G.C. Chromophore formation in green fluorescent protein. Biochemistry 36, 6786–6791 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Zimmer, M. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem. Rev. 102, 759–781 (2002).

    Article  CAS  Google Scholar 

  8. Demidov, V.V. et al. Fast complementation of split fluorescent protein triggered by DNA hybridization. Proc. Natl. Acad. Sci. USA 103, 2052–2056 (2006).

    Article  CAS  Google Scholar 

  9. Endoh, T., Funabashi, H., Mie, M. & Kobatake, E. Method for detection of specific nucleic acids by recombinant protein with fluorescent resonance energy transfer. Anal. Chem. 77, 4308–4314 (2005).

    Article  CAS  Google Scholar 

  10. Marras, S.A., Kramer, F.R. & Tyagi, S. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30, e122 (2002).

    Article  Google Scholar 

  11. Tan, W., Wang, K. & Drake, T.J. Molecular beacons. Curr. Opin. Chem. Biol. 8, 547–553 (2004).

    Article  CAS  Google Scholar 

  12. Sambrook, J.F. & Russell, D.W. (eds.) Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, 2001).

  13. Hamad-Schifferli, K., Schwartz, J.J., Santos, A.T., Zhang, S. & Jacobson, J.M. Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature 415, 152–155 (2002).

    Article  Google Scholar 

  14. Sekar, M.M., Bloch, W. & St. John, P.M. Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres. Nucleic Acids Res. 33, 366–375 (2005).

    Article  CAS  Google Scholar 

  15. Hu, C.D. & Kerppola, T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 21, 539–545 (2003).

    Article  CAS  Google Scholar 

  16. Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23, 605–613 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge our graduate students P. Chalasani and H.-W. Yiu, as well as C. Witte-Hoffmann, I. Smolina and Y. Yu, who participated in the protocol development. We also thank C.R. Cantor for inspiration and encouragement in this project.

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  1. Center for Advanced Biotechnology, Boston University, 36 Cummington Street, Boston, 02215, Massachusetts, USA

    Vadim V Demidov & Natalia E Broude

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  1. Vadim V Demidov
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  2. Natalia E Broude
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Correspondence to Vadim V Demidov or Natalia E Broude.

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The authors declare no competing financial interests.

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Demidov, V., Broude, N. Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro. Nat Protoc 1, 714–719 (2006). https://doi.org/10.1038/nprot.2006.114

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