Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression.
Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression.
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DOI：
10.1371/journal.pone.0174689
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Liu AP
中科院分区：
文献类型：
--
作者：
Ho KK;Lee JW;Durand G;Majumder S;Liu AP
人工细胞模型的发展需要将生物分子封装在膜结合的隔室中。使用哺乳动物无细胞表达（CFE）系统作为人造细胞的“细胞质”的研究有限。我们利用玻璃毛细管微流体在双乳液模板囊泡内封装哺乳动物CFE。与封装简单的缓冲溶液相比，无HeLa细胞裂解物的物理化学性质的复杂性提出了挑战。特别是，我们发现包裹哺乳动物HeLa CFE的双乳液模板囊泡形成聚集体，但细菌CFE没有形成聚集体。聚集体不是由由CFE制成的蛋白质的不溶性引起的，也不是由于哺乳动物CFE与双乳中间相的有机溶剂的相互作用。我们发现，聚乙烯醇（PVA）表面活性剂（一种关键的双乳液稳定表面活性剂）的浓度和哺乳动物CFE裂解物的浓度决定了这种聚集。尽管囊泡不稳定和蛋白质表达减少，我们通过封装哺乳动物CFE系统证明了蛋白质表达。通过质谱分析和Western blot，我们鉴定并验证了肌动蛋白是哺乳动物CFE中与PVA表面活性剂聚集的蛋白之一。我们的工作建立了一个基线描述，哺乳动物CFE系统封装在双乳液模板囊泡中，作为构建人工细胞的平台。
Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the ‘cytosol’ of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells.