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Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Nature Biotechnology volume 23, pages 102–107 (2005)Cite this article

Abstract

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility1,2,3,4 or may not work in living cells5,6,7. Green fluorescent protein (GFP) fusions can misfold8 or exhibit altered processing9. Fluorogenic biarsenical FLaSH or ReASH10 substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization10. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP11, but the GFP fragments reported thus far are large and fold poorly11,12, require chemical ligation13 or fused interacting partners to force their association11,12,13,14, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP11,12. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.

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Figure 1: Principle of split GFP complementation and engineering fragments for improved solubility and complementation.
Figure 2: In vitro characterization of split GFP complementation reaction.
Figure 3: In vitro protein quantification and in vivo protein expression and solubility screens.

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References

  1. Ullmann, A., Jacob, F. & Monod, J. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24, 339–343 (1967).

    Article  CAS  Google Scholar 

  2. Wehrman, T., Kleaveland, B., Her, J.H., Balint, R.F. & Blau, H.M. Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments. Proc. Natl. Acad. Sci. USA 99, 3469–3474 (2002).

    Article  CAS  Google Scholar 

  3. Nixon, A.E. & Benkovic, S.J. Improvement in the efficiency of formyl transfer of a GAR transformylase hybrid enzyme. Protein Eng. 13, 323–327 (2000).

    Article  CAS  Google Scholar 

  4. Wigley, W.C., Stidham, R.D., Smith, N.M., Hunt, J.F. & Thomas, P.J. Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein. Nat. Biotechnol. 19, 131–136 (2001).

    Article  CAS  Google Scholar 

  5. Kim, J.S. & Raines, R.T. Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci. 2, 348–356 (1993).

    Article  CAS  Google Scholar 

  6. Kelemen, B.R. et al. Hypersensitive substrate for ribonucleases. Nucleic Acids Res. 27, 3696–3701 (1999).

    Article  CAS  Google Scholar 

  7. Richards, F.M. & Vithayathil, P.J. The preparation of subtilisn-modified ribonuclease and the separation of the peptide and protein components. J. Biol. Chem. 234, 1459–1465 (1959).

    CAS  PubMed  Google Scholar 

  8. Waldo, G.S., Standish, B.M., Berendzen, J. & Terwilliger, T.C. Rapid proteinfolding assay using green fluorescent protein. Nat. Biotechnol. 17, 691–695 (1999).

    Article  CAS  Google Scholar 

  9. Bertens, P., Heijne, W., Van Der Wel, N., Wellink, J. & Van Kammen, A. Studies on the C-terminus of the Cowpea mosaic virus movement protein. Arch. Virol. 148, 265–279 (2003).

    Article  CAS  Google Scholar 

  10. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  11. 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 

  12. 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 

  13. Ozawa, T. & Umezawa, Y. Detection of protein-protein interactions in vivo based on protein splicing. Curr. Opin. Chem. Biol. 5, 578–583 (2001).

    Article  CAS  Google Scholar 

  14. Ozawa, T., Takeuchi, T.M., Kaihara, A., Sato, M. & Umezawa, Y. Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal. Chem. 73, 5866–5874 (2001).

    Article  CAS  Google Scholar 

  15. Crameri, A., Whitehorn, E.A., Tate, E. & Stemmer, W.P. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319 (1996).

    Article  CAS  Google Scholar 

  16. Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

    Article  CAS  Google Scholar 

  17. Fitz-Gibbon, S. et al. A fosmid-based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum. Extremophiles 1, 36–51 (1997).

    Article  CAS  Google Scholar 

  18. Stemmer, W.P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Tal, M., Silberstein, A. & Nusser, E. Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer. J. Biol. Chem. 260, 9976–9980 (1985).

    CAS  PubMed  Google Scholar 

  21. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89 (1990).

    Article  CAS  Google Scholar 

  22. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  Google Scholar 

  23. Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421 (1999).

    Article  CAS  Google Scholar 

  24. Fahnert, B., Lilie, H. & Neubauer, P. Inclusion bodies: formation and utilisation. Adv. Biochem. Eng. Biotechnol. 89, 93–142 (2004).

    CAS  PubMed  Google Scholar 

  25. Gerstein, M., Edwards, A., Arrowsmith, C.H. & Montelione, G.T. Structural genomics: current progress. Science 299, 1663 (2003).

    Article  CAS  Google Scholar 

  26. Makrides, S.C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538 (1996).

    Article  CAS  Google Scholar 

  27. Yokoyama, S. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem. Biol. 7, 39–43 (2003).

    Article  CAS  Google Scholar 

  28. Fox, J.D., Kapust, R.B. & Waugh, D.S. Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins. Protein Sci. 10, 622–630 (2001).

    Article  CAS  Google Scholar 

  29. Pelletier, J.N., Campbell-Valois, F.X. & Michnick, S.W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).

    Article  CAS  Google Scholar 

  30. Pelletier, J.N., Arndt, K.M., Pluckthun, A. & Michnick, S.W. An in vivo library-versus-library selection of optimized protein-protein interactions. Nat. Biotechnol. 17, 683–690 (1999).

    Article  CAS  Google Scholar 

  31. Goh, C.S. et al. Mining the structural genomics pipeline: identification of protein properties that affect high-throughput experimental analysis. J. Mol. Biol. 336, 115–130 (2004).

    Article  CAS  Google Scholar 

  32. Terwilliger, T.C. Structures and technology for biologists. Nat. Struct. Mol. Biol. 11, 296–297 (2004).

    Article  CAS  Google Scholar 

  33. Armstrong, N., de Lencastre, A. & Gouaux, E. A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci. 8, 1475–1483 (1999).

    Article  CAS  Google Scholar 

  34. Waldo, G.S. Genetic screens and directed evolution for protein solubility. Curr. Opin. Chem. Biol. 7, 33–38 (2003).

    Article  CAS  Google Scholar 

  35. Knaust, R.K. & Nordlund, P. Screening for soluble expression of recombinant proteins in a 96-well format. Anal. Biochem. 297, 79–85 (2001).

    Article  CAS  Google Scholar 

  36. Ozawa, T., Sako, Y., Sato, M., Kitamura, T. & Umezawa, Y. A genetic approach to identifying mitochondrial proteins. Nat. Biotechnol. 21, 287–293 (2003).

    Article  CAS  Google Scholar 

  37. Zhang, S., Ma, C. & Chalfie, M. Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119, 137–144 (2004).

    Article  CAS  Google Scholar 

  38. Morris, M.C., Depollier, J., Mery, J., Heitz, F. & Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173–1176 (2001).

    Article  CAS  Google Scholar 

  39. 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 

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Acknowledgements

The authors wish to acknowledge B. Mark and J.-D. Pédelacq for helpful comments and the National Institutes of Health and Laboratory-directed research and development program for generous support.

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Authors and Affiliations

  1. Bioscience Division, MS-M888, Los Alamos National Laboratory, PO Box 1663, Los Alamos, 87545, New Mexico, USA

    Stéphanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo

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  1. Stéphanie Cabantous
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  2. Thomas C Terwilliger
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  3. Geoffrey S Waldo
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Corresponding author

Correspondence to Geoffrey S Waldo.

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Competing interests

The authors are inventors named in a utility patent (Self-assembling fragments of GFP) submitted to the USPTO by Los Alamos National Laboratories on behalf of the University of California.

Supplementary information

Supplementary Fig. 1

Map of pTET-SpecR plasmid. (PDF 32 kb)

Supplementary Fig. 2

Sequential of co-induction of BL21(DE3) containing compatible plasmids expressing GFP 1–10 variants, and sulfite reductase fused to GFP 11 wild type. (PDF 36 kb)

Supplementary Fig. 3

Effect of Urea on the complementation reaction. (PDF 24 kb)

Supplementary Fig. 4

pH dependence of complementation (PDF 88 kb)

Supplementary Table 1

Amino acid sequence of the GFP 11 mutants (PDF 20 kb)

Supplementary Table 2

Comparison of the whole-cell fluorescence of pre-induction and post-induction GFP expression using the pTET-SpecR vector or pPROTET CmR commercial vector (Clontech). (PDF 14 kb)

Supplementary Methods (PDF 20 kb)

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Cabantous, S., Terwilliger, T. & Waldo, G. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol 23, 102–107 (2005). https://doi.org/10.1038/nbt1044

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