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.

  • Technical Report
  • Published:

A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography

Nature Medicine volume 16, pages 921–926 (2010)Cite this article

Subjects

Abstract

Improved techniques to noninvasively image protein-protein interactions (PPIs) are essential. We molecularly engineered a positron emission tomography (PET)-based split reporter (herpes simplex virus type 1 thymidine kinase), cleaved between Thr265 and Ala266, and used this in a protein-fragment complementation assay (PCA) to quantify PPIs in mammalian cells and to microPET image them in living mice. An introduced point mutation (V119C) markedly enhanced thymidine kinase complementation in PCAs, on the basis of rapamycin modulation of FKBP12-rapamycin-binding domain (FRB) and FKBP12 (FK506 binding protein), the interaction of hypoxia-inducible factor-1α with the von Hippel-Lindau tumor suppressor, and in an estrogen receptor intramolecular protein folding assay. Applications of this unique split thymidine kinase are potentially far reaching, including, for example, considerably more accurate monitoring of immune and stem cell therapies, allowing for fully quantitative and tomographic PET localization of PPIs in preclinical small- and large-animal models of disease.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Principle of the thymidine kinase PCA, expression vectors used and evaluation of plasmid vector constructs containing the V119C mutation.
Figure 2: Subcellular localization of components of the PCA.
Figure 3: Testing the thymidine kinase PCA in separate cell lines and PPI systems.
Figure 4: Imaging of tumors containing the split thymidine kinase constructs.
Figure 5: The relative advantage of PET over optical imaging in imaging sources of signal at depths below 1 cm from the exterior is exemplified in this experiment.

Similar content being viewed by others

References

  1. Valdar, W.S. & Thornton, J.M. Protein-protein interfaces: analysis of amino acid conservation in homodimers. Proteins 42, 108–124 (2001).

    Article  CAS  Google Scholar 

  2. Shoemaker, B.A. & Panchenko, A.R. Deciphering protein-protein interactions. Part I. Experimental techniques and databases. PLoS Comput. Biol. 3, e42 (2007).

    Article  Google Scholar 

  3. Massoud, T.F. & Gambhir, S.S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 17, 545–580 (2003).

    Article  CAS  Google Scholar 

  4. Michnick, S.W. Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments. Curr. Opin. Struct. Biol. 11, 472–477 (2001).

    Article  CAS  Google Scholar 

  5. 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 

  6. Michnick, S.W., Remy, I., Campbell-Valois, F.X., Vallee-Belisle, A. & Pelletier, J.N. Detection of protein-protein interactions by protein fragment complementation strategies. Methods Enzymol. 328, 208–230 (2000).

    Article  CAS  Google Scholar 

  7. Park, K. et al. A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system. Protein J. 26, 107–116 (2007).

    Article  CAS  Google Scholar 

  8. 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 

  9. Paulmurugan, R., Umezawa, Y. & Gambhir, S.S. Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc. Natl. Acad. Sci. USA 99, 15608–15613 (2002).

    Article  CAS  Google Scholar 

  10. Paulmurugan, R. & Gambhir, S.S. Monitoring protein-protein interactions using split synthetic Renilla luciferase protein fragment–assisted complementation. Anal. Chem. 75, 1584–1589 (2003).

    Article  CAS  Google Scholar 

  11. Tjuvajev, J.G. et al. Imaging the expression of transfected genes in vivo. Cancer Res. 55, 6126–6132 (1995).

    CAS  PubMed  Google Scholar 

  12. Kang, K.W., Min, J.J., Chen, X. & Gambhir, S.S. Comparison of [14C]FMAU, [3H]FEAU, [14C]FIAU, and [3H]PCV for monitoring reporter gene expression of wild type and mutant herpes simplex virus type 1 thymidine kinase in cell culture. Mol. Imaging Biol. 7, 296–303 (2005).

    Article  Google Scholar 

  13. Iyer, M. et al. 8-[18F]Fluoropenciclovir: an improved reporter probe for imaging HSV1-tk reporter gene expression in vivo using PET. J. Nucl. Med. 42, 96–105 (2001).

    CAS  PubMed  Google Scholar 

  14. Gambhir, S.S. et al. A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography. Proc. Natl. Acad. Sci. USA 97, 2785–2790 (2000).

    Article  CAS  Google Scholar 

  15. Massoud, T.F. Novel Approaches to Molecular Imaging of Protein-Protein Interactions in Living Subjects. PhD thesis, Univ. Cambridge (2007).

    Google Scholar 

  16. Spotts, J.M., Dolmetsch, R.E. & Greenberg, M.E. Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 15142–15147 (2002).

    Article  CAS  Google Scholar 

  17. Chen, J. & Schreiber, S.L. Identification of an 11-kDa FKBP12-rapamycin–binding domain within the 289-kDa FKBP12-rapamycin–associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. USA 92, 4947–4951 (1995).

    Article  CAS  Google Scholar 

  18. Remy, I. & Michnick, S.W. Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays. Proc. Natl. Acad. Sci. USA 96, 5394–5399 (1999).

    Article  CAS  Google Scholar 

  19. Wurth, C., Thomas, R.M., Folkers, G. & Scapozza, L. Folding and self-assembly of herpes simplex virus type 1 thymidine kinase. J. Mol. Biol. 313, 657–670 (2001).

    Article  CAS  Google Scholar 

  20. Jaakkola, P. et al. Targeting HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468–472 (2001).

    Article  CAS  Google Scholar 

  21. Paulmurugan, R. & Gambhir, S.S. An intramolecular folding sensor for imaging estrogen receptor-ligand interactions. Proc. Natl. Acad. Sci. USA 103, 15883–15888 (2006).

    Article  CAS  Google Scholar 

  22. Peñuelas, I. et al. Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients. Gastroenterology 128, 1787–1795 (2005).

    Article  Google Scholar 

  23. Brannigan, J.A. & Wilkinson, A.J. Protein engineering 20 years on. Nat. Rev. Mol. Cell Biol. 3, 964–970 (2002).

    Article  CAS  Google Scholar 

  24. Chen, R. Enzyme engineering: rational redesign versus directed evolution. Trends Biotechnol. 19, 13–14 (2001).

    Article  CAS  Google Scholar 

  25. Massoud, T.F. & Gambhir, S.S. Integrating noninvasive molecular imaging into molecular medicine: an evolving paradigm. Trends Mol. Med. 13, 183–191 (2007).

    Article  CAS  Google Scholar 

  26. Michnick, S.W., Ear, P.H., Manderson, E.N., Remy, I. & Stefan, E. Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat. Rev. Drug Discov. 6, 569–582 (2007).

    Article  CAS  Google Scholar 

  27. Qi, J., Leahy, R.M., Cherry, S.R., Chatziioannou, A. & Farquhar, T.H. High-resolution 3D Bayesian image reconstruction using the microPET small-animal scanner. Phys. Med. Biol. 43, 1001–1013 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Gobalakrishnan, J. Willmann and O. Gheysens for their assistance with the microPET imaging. T. Massoud was supported in part by US National Cancer Institute In Vivo Cellular and Molecular Imaging Center grant P50 CA86306 and by a Mid-Career Award for Established Practitioners from The Health Foundation, General Electric Medical Systems, the Palgrave Brown Foundation, the Cancer Prevention Research Trust, a Royal College of Radiologists X-Appeal pump priming grant, the Steel Charitable Trust, the Sir Samuel Scott of Yews Trust and the National Institute of Health Research Cambridge Biomedical Research Centre, all in the UK. This work was funded by US National Cancer Institute grant In Vivo Cellular and Molecular Imaging Center P50 CA114747 (S.S.G.) and US National Cancer Institute RO1 CA082214 (S.S.G.).

Author information

Authors and Affiliations

  1. Department of Radiology, University of Cambridge School of Clinical Medicine, Cambridge, UK

    Tarik F Massoud

  2. Department of Oncology, University of Cambridge School of Clinical Medicine, Cambridge, UK

    Tarik F Massoud

  3. Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Stanford, California, USA

    Tarik F Massoud, Ramasamy Paulmurugan & Sanjiv S Gambhir

  4. Department of Radiology, Stanford University School of Medicine, Stanford, California, USA

    Tarik F Massoud, Ramasamy Paulmurugan & Sanjiv S Gambhir

  5. Department of Bioengineering, Stanford University School of Medicine, Stanford, California, USA

    Sanjiv S Gambhir

Authors
  1. Tarik F Massoud
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ramasamy Paulmurugan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sanjiv S Gambhir
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors designed the experiments. S.S.G. supervised the experiments. T.F.M. and R.P. performed the experiments. T.F.M. and S.S.G. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Sanjiv S Gambhir.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Discussion, Supplementary Figures 1–19 and Supplementary Tables 1 and 2 (PDF 1181 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Massoud, T., Paulmurugan, R. & Gambhir, S. A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography. Nat Med 16, 921–926 (2010). https://doi.org/10.1038/nm.2185

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2185

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research