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

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.

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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.

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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.).

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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.

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Correspondence to Sanjiv S Gambhir.

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The authors declare no competing financial interests.

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

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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

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