Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation,ACS Synthetic Biology

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Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-11-07 00:00:00 , DOI: 10.1021/acssynbio.7b00325
Wenting Zhao ₁ , Lara Pferdehirt ₂ , Laura Segatori _{1,

2,

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Affiliation

Protein function is typically studied and engineered by modulating protein levels within the complex cellular environment. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, we developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the Camelidae species heavy-chain antibody (nanobody) fused to a degron signal that mediates degradation through the proteasome. We provide proof-of-principle demonstration of targeted degradation using a nanobody against the green fluorescent protein (GFP). Guided by predictive modeling, we show that customizing the NanoDeg rate of synthesis, rate of degradation, and mode of degradation enables quantitative and predictable control over the target’s levels. Integrating the GFP-specific NanoDeg within a genetic circuit based on stimulus-dependent GFP output results in enhanced dynamic range and resolution of the output signal. By providing predictable control over cellular proteins’ levels, the NanoDeg system could be readily used for a variety of systems-level analyses of cellular protein function.

中文翻译：

通过蛋白酶体降解定量预测细胞蛋白水平

通常通过调节复杂细胞环境中的蛋白质水平来研究和设计蛋白质功能。为了实现对细胞蛋白质水平的快速，有针对性和可预测的控制，而无需对目标进行遗传操作，我们开发了一种基于双功能分子（NanoDeg）的翻译后耗竭技术，该双功能分子由骆驼科动物的抗原结合片段组成物种重链抗体（纳米抗体）融合到介导通过蛋白酶体降解的地龙信号上。我们提供针对使用绿色荧光蛋白（GFP）的纳米抗体进行靶向降解的原理证明。在预测建模的指导下，我们表明定制NanoDeg的合成速率，降解速率和降解模式可以对目标水平进行定量和可预测的控制。基于依赖于刺激的GFP输出，将GFP特定的NanoDeg整合到遗传电路中，可提高输出信号的动态范围和分辨率。通过提供对细胞蛋白质水平的可预测控制，NanoDeg系统可轻松用于细胞蛋白质功能的各种系统级分析。

更新日期：2017-11-08

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