A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography.
A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography.
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非侵入性成像蛋白质-蛋白质相互作用（PPI）的改进技术至关重要。我们分子设计了一种基于正电子发射断层扫描（PET）的分裂报告基因（单纯疱疹病毒1型胸苷激酶[TK]），在Thr 265和Ala 266之间分裂，并将其用于蛋白质片段互补试验（PCA）中，以定量测量哺乳动物细胞中的PPI，并在活体小鼠中对其进行microPET成像。引入的点突变（V119 C）显著增强了PCA中的TK互补，这是基于雷帕霉素对FRB（FKBP 12-雷帕霉素结合结构域）和FKBP 12（FK 506结合蛋白）的调节、低氧诱导因子-1 α与von Hippel-Lindau肿瘤抑制因子的相互作用以及雌激素受体分子内蛋白折叠试验。这种新型裂解TK的应用可能意义深远，包括例如对免疫和干细胞治疗进行更准确的监测，允许在临床前小型和大型动物疾病模型中对PPI进行前所未有的完全定量和断层扫描PET定位。
Improved techniques to non-invasively 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 [TK]), split between Thr265 and Ala266, and used this in a protein-fragment complementation assay (PCA) to quantitatively measure PPIs in mammalian cells and to microPET image them in living mice. An introduced point mutation (V119C) significantly enhanced TK complementation in PCAs based on rapamycin modulation of FRB (FKBP12-rapamycin-binding domain) and FKBP12 (FK506 binding protein), on interaction of hypoxia-inducible factor-1α and the von Hippel-Lindau tumor suppressor, and in an estrogen receptor intramolecular protein folding assay. Applications of this novel split TK are potentially far-reaching, including for example considerably more accurate monitoring of immune and stem cell therapies, allowing unprecedented fully quantitative and tomographic PET localization of PPIs in pre-clinical small and large animal models of disease.