Phase Separation and Protein Partitioning in Compartmentalized Cell-Free Expression Reactions
Phase Separation and Protein Partitioning in Compartmentalized Cell-Free Expression Reactions
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DOI：
10.1021/acs.biomac.1c00546
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发表时间：
2021-07-14
影响因子：
6.2
通讯作者：
Maeda, Yusuke T.
中科院分区：
文献类型：
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作者：
Kato, Shuzo;Garenne, David;Maeda, Yusuke T.
液-液相分离（LLPS）对于在细胞大小的空间中控制从基因表达到蛋白质降解的广泛反应是重要的。为了更好地理解这种相分离结构与蛋白质合成的相容性，我们研究了无细胞转录-翻译（TXTL）反应中的紧急LLPS。当由许多蛋白质组成的TXTL反应被浓缩时，均匀混合的状态变得不稳定，并且自发地形成无膜相。当TXTL反应被封闭在油包水乳液液滴中时，诱导这种LLPS液滴形成，其中水从表面蒸发。当乳液液滴收缩时，较小的LLPS液滴出现在乳液液滴内，并合并成大的相分离结构域，该相分离结构域划分合成的报告蛋白的定位。PEG在TXTL反应中的存在不仅对于通用的无细胞蛋白质合成很重要，而且对于能够进行蛋白质分配的两个大结构域的形成也很重要。我们的研究结果可能揭示了LLPS形成和无细胞蛋白质合成对合成细胞器的建设的动态相互作用。
Liquid-liquid phase separation (LLPS) is important to control a wide range of reactions from gene expression to protein degradation in a cell-sized space. To bring a better understanding of the compatibility of such phase-separated structures with protein synthesis, we study emergent LLPS in a cell-free transcription- translation (TXTL) reaction. When the TXTL reaction composed of many proteins is concentrated, the uniformly mixed state becomes unstable, and membrane-less phases form spontaneously. This LLPS droplet formation is induced when the TXTL reaction is enclosed in water-in-oil emulsion droplets, in which water evaporates from the surface. As the emulsion droplets shrink, smaller LLPS droplets appear inside the emulsion droplets and coalesce into large phase-separated domains that partition the localization of synthesized reporter proteins. The presence of PEG in the TXTL reaction is important not only for versatile cell-free protein synthesis but also for the formation of two large domains capable of protein partitioning. Our results may shed light on the dynamic interplay of LLPS formation and cell-free protein synthesis toward the construction of synthetic organelles.