Ahn So Yeon, Obermeyer Allie C
Department of Chemical Engineering, Columbia University, New York, NY.
bioRxiv. 2024 Apr 3:2024.04.02.587643. doi: 10.1101/2024.04.02.587643.
Liquid-liquid phase separation of biomolecules is increasingly recognized as 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 has 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 multi-component mixture. The behavior of individual proteins was evaluated using a defined mixture of proteins that mimics the charge profile of the proteome. To allow for 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 the coacervation behavior of individual proteins in the mixture was consistent with each other, which was distinctive from the behavior when each protein was evaluated in a single-protein system. Subtle differences in biophysical properties between the proteins 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.
生物分子的液-液相分离与各种细胞功能的相关性日益受到认可，生物大分子（尤其是蛋白质）的复合凝聚正成为这一现象的关键机制。由于其潜在的可扩展性、水相操作以及产生高浓缩产物的能力，复合凝聚也正在作为一种潜在的蛋白质纯化方法进行探索。然而，迄今为止，大多数复合凝聚研究都评估了两种带相反电荷的大分子二元混合物的相行为。因此，尚未建立对复杂生物混合物相行为的全面理解。为了解决这个问题，设计了一组工程蛋白，以便对多组分混合物中单个蛋白质的复合凝聚进行定量分析。使用模拟蛋白质组电荷分布的特定蛋白质混合物评估单个蛋白质的行为。为了直接定量每个相中蛋白质，使用光谱分离的荧光蛋白构建蛋白质混合物。通过这种定量分析，我们观察到混合物中单个蛋白质的凝聚行为彼此一致，这与在单蛋白系统中评估每个蛋白质时的行为不同。蛋白质之间生物物理性质的细微差异在混合物中变得明显，这使我们能够阐明蛋白质复合凝聚的参数。基于这一认识，我们成功设计了从蛋白质混合物中富集一系列目标蛋白质的方法。
