Kapelner Rachel A, Obermeyer Allie C
Department of Chemical Engineering , Columbia University , New York , NY 10027 , USA . Email:
Chem Sci. 2019 Jan 17;10(9):2700-2707. doi: 10.1039/c8sc04253e. eCollection 2019 Mar 7.
Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. The protein design factors that govern complex coacervation under varying conditions are still relatively unexplored. Recent work indicates that proteins with an intrinsically disordered region, a higher net charge, or a patch of charged residues are more likely to undergo a phase transition. Based on these design parameters, polyionic coacervation tags were designed and assessed for their ability to promote protein complex coacervation with oppositely charged polyelectrolytes. The phase behavior of a panel of engineered proteins was evaluated with the strong polycation poly(4-vinyl -methyl pyridinium iodide). Proteins containing the ionic tags formed liquid coacervate droplets, while isotropically charged protein variants formed solid precipitates. The ionic tags also promoted phase separation at higher salt concentrations than an isotropic distribution of charge on the protein surface. The salt dependence of the protein complex coacervation could be predicted independently for tagged or isotropic variants by the ratio of negative-to-positive residues on the proteins and universally by calculating the distance between like charges. The addition of just a six residue polyionic tag generated a globular protein capable of liquid-liquid phase separation at physiological pH and ionic strength. This model system has provided the initial demonstration that short, ionic polypeptide sequences (6-18 amino acids) can drive the liquid-liquid phase separation of globular proteins.
水溶液中带相反电荷的聚电解质可发生称为复合凝聚的液-液相分离。两性蛋白质与带相反电荷的聚电解质的复合凝聚越来越受到关注，因为它会形成富含蛋白质的相，在蛋白质治疗、蛋白质纯化和生物催化方面具有潜在应用。然而，许多球状蛋白质与带相反电荷的聚电解质混合时不会发生相分离，而那些确实发生相分离的蛋白质是在狭窄的浓度、pH值和离子强度范围内进行的。在不同条件下控制复合凝聚的蛋白质设计因素仍相对未被探索。最近的研究表明，具有内在无序区域、较高净电荷或一片带电荷残基的蛋白质更有可能发生相变。基于这些设计参数，设计了聚离子凝聚标签，并评估了它们促进蛋白质与带相反电荷的聚电解质复合凝聚的能力。用强聚阳离子聚（4-乙烯基-甲基碘化吡啶）评估了一组工程蛋白的相行为。含有离子标签的蛋白质形成了液体凝聚液滴，而各向同性带电的蛋白质变体形成了固体沉淀。与蛋白质表面电荷的各向同性分布相比，离子标签还能在更高盐浓度下促进相分离。通过蛋白质上负残基与正残基的比例，可以独立预测标记或各向同性变体的蛋白质复合凝聚对盐的依赖性，并且通过计算同性电荷之间的距离可以普遍预测。仅添加一个六个残基的聚离子标签就产生了一种能够在生理pH值和离子强度下进行液-液相分离的球状蛋白质。这个模型系统初步证明了短的离子多肽序列（6-18个氨基酸）可以驱动球状蛋白质的液-液相分离。
