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A Split Transcriptional Repressor That Links Protein Solubility to an Orthogonal Genetic Circuit
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-08-08 00:00:00 , DOI: 10.1021/acssynbio.8b00129 Yimeng Zeng 1 , Alicia M Jones 2 , Emily E Thomas 2 , Barbara Nassif 2 , Jonathan J Silberg 2, 3 , Laura Segatori 1, 2, 3
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-08-08 00:00:00 , DOI: 10.1021/acssynbio.8b00129 Yimeng Zeng 1 , Alicia M Jones 2 , Emily E Thomas 2 , Barbara Nassif 2 , Jonathan J Silberg 2, 3 , Laura Segatori 1, 2, 3
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Monitoring the aggregation of proteins within the cellular environment is key to investigating the molecular mechanisms underlying the formation of off-pathway protein assemblies associated with the development of disease and testing therapeutic strategies to prevent the accumulation of non-native conformations. It remains challenging, however, to couple protein aggregation events underlying the cellular pathogenesis of a disease to genetic circuits and monitor their progression in a quantitative fashion using synthetic biology tools. To link the aggregation propensity of a target protein to the expression of an easily detectable reporter, we investigated the use of a transcriptional AND gate system based on complementation of a split transcription factor. We first identified two-fragment tetracycline repressor (TetR) variants that can be regulated via ligand-dependent induction and demonstrated that split TetR variants can function as transcriptional AND gates in both bacteria and mammalian cells. We then adapted split TetR for use as an aggregation sensor. Protein aggregation was detected by monitoring complementation between a larger TetR fragment that serves as a “detector” and a smaller TetR fragment expressed as a fusion to an aggregation-prone protein that serves as a “sensor” of the target protein aggregation status. This split TetR represents a novel genetic component that can be used for a wide range of applications in bacterial as well as mammalian synthetic biology and a much needed cell-based sensor for monitoring a protein’s conformational status in complex cellular environments.
中文翻译：
将蛋白质溶解度与正交遗传电路联系起来的分裂转录抑制子
监测细胞环境内蛋白质的聚集是研究与疾病发展相关的旁路蛋白质组装体形成的分子机制以及测试防止非天然构象积累的治疗策略的关键。然而，将疾病细胞发病机制背后的蛋白质聚集事件与遗传回路结合起来，并使用合成生物学工具以定量方式监测其进展仍然具有挑战性。为了将目标蛋白的聚集倾向与易于检测的报告基因的表达联系起来，我们研究了基于分裂转录因子互补的转录与门系统的使用。我们首先鉴定了可以通过配体依赖性诱导调节的双片段四环素阻遏物（TetR）变体，并证明分裂的 TetR 变体可以在细菌和哺乳动物细胞中充当转录与门。然后我们调整 split TetR 用作聚合传感器。通过监测充当“检测器”的较大 TetR 片段和表达为与易于聚集的蛋白质融合的较小 TetR 片段（充当目标蛋白质聚集状态的“传感器”）之间的互补性来检测蛋白质聚集。这种分裂的 TetR 代表了一种新型遗传成分，可在细菌和哺乳动物合成生物学中广泛应用，也是一种急需的基于细胞的传感器，用于监测复杂细胞环境中蛋白质的构象状态。
更新日期：2018-08-08
中文翻译：
将蛋白质溶解度与正交遗传电路联系起来的分裂转录抑制子
监测细胞环境内蛋白质的聚集是研究与疾病发展相关的旁路蛋白质组装体形成的分子机制以及测试防止非天然构象积累的治疗策略的关键。然而，将疾病细胞发病机制背后的蛋白质聚集事件与遗传回路结合起来，并使用合成生物学工具以定量方式监测其进展仍然具有挑战性。为了将目标蛋白的聚集倾向与易于检测的报告基因的表达联系起来，我们研究了基于分裂转录因子互补的转录与门系统的使用。我们首先鉴定了可以通过配体依赖性诱导调节的双片段四环素阻遏物（TetR）变体，并证明分裂的 TetR 变体可以在细菌和哺乳动物细胞中充当转录与门。然后我们调整 split TetR 用作聚合传感器。通过监测充当“检测器”的较大 TetR 片段和表达为与易于聚集的蛋白质融合的较小 TetR 片段（充当目标蛋白质聚集状态的“传感器”）之间的互补性来检测蛋白质聚集。这种分裂的 TetR 代表了一种新型遗传成分，可在细菌和哺乳动物合成生物学中广泛应用，也是一种急需的基于细胞的传感器，用于监测复杂细胞环境中蛋白质的构象状态。
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