A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography.
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-1alpha 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.
Massoud
Tarik F
TF
Department of Radiology, University of Cambridge School of Clinical Medicine, Cambridge, UK.
Paulmurugan
Ramasamy
R
Gambhir
Sanjiv S
SS
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D017930:Genes, Reporter / Q000502:physiology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000502:physiology; D049268:Positron-Emission Tomography / Q000379:methods; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000378:metabolism; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014183:Transplantation, Heterologous; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010446:Peptide Fragments; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D013937:Thymidine Kinase; D022061:Tacrolimus Binding Protein 1A; D022021:Tacrolimus Binding Proteins