Selectivity of Complex Coacervation in Multi-Protein Mixtures.
Selectivity of Complex Coacervation in Multi-Protein Mixtures.
复制标题
多蛋白质混合物中复杂凝聚的选择性。
DOI：
10.1101/2024.04.02.587643
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Obermeyer,AllieC
中科院分区：
文献类型：
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作者：
Ahn,SoYeon;Obermeyer,AllieC
生物分子的液-液相分离越来越多地被认为与各种细胞功能相关，生物大分子，特别是蛋白质的复合凝聚正在成为这种现象的关键机制。由于其潜在的可扩展性、水性操作和生产高度浓缩产品的能力，复合凝聚也被探索为潜在的蛋白质纯化方法。然而，迄今为止，大多数复凝聚的研究已经评估了两个相反电荷的大分子的二元混合物的相行为。因此，对复杂生物混合物的相行为的全面理解尚未建立。为了解决这个问题，设计了一组工程蛋白质，以允许定量分析多组分混合物中单个蛋白质的复合凝聚。单个蛋白质的行为是用一种确定的蛋白质混合物来评价的，这种混合物模拟了大肠杆菌蛋白质组的电荷分布。为了允许直接定量每个相中的蛋白质，使用光谱分离的荧光蛋白来构建蛋白质混合物。从该定量分析中，我们观察到蛋白质凝聚在混合物中是同步的，这与在单蛋白质系统中评价每种蛋白质时的行为不同。蛋白质之间的生物物理性质的微妙差异，如个别带电残基和整体电荷密度的电离，在混合物中变得明显，这使我们能够阐明蛋白质复合物凝聚的参数。有了这样的理解，我们成功地设计了从蛋白质混合物中富集一系列感兴趣的蛋白质的方法。
Liquid–liquid phase separation of biomolecules is increasingly recognized as being relevant to various cellular functions, and complex coacervation of biomacromolecules, particularly proteins, is emerging as a key mechanism for this phenomenon. Complex coacervation is also being explored as a potential protein purification method due to its potential scalability, aqueous operation, and ability to produce a highly concentrated product. However, to date, most studies of complex coacervation have evaluated the phase behavior of a binary mixture of two oppositely charged macromolecules. Therefore, a comprehensive understanding of the phase behavior of complex biological mixtures is yet to be established. To address this, a panel of engineered proteins was designed to allow for quantitative analysis of the complex coacervation of individual proteins within a multicomponent mixture. The behavior of individual proteins was evaluated using a defined mixture of proteins that mimics the charge profile of theEscherichia coliproteome. To allow for the direct quantification of proteins in each phase, spectrally separated fluorescent proteins were used to construct the protein mixture. From this quantitative analysis, we observed that protein coacervation was synchronized in the mixture, which was distinctive from the behavior when each protein was evaluated in a single-protein system. Subtle differences in biophysical properties between the proteins, such as the ionization of individual charged residues and overall charge density, became noticeable in the mixture, which allowed us to elucidate parameters for protein complex coacervation. With this understanding, we successfully designed methods to enrich a range of proteins of interest from a mixture of proteins.