A mechanistic examination of salting out in protein–polymer membrane interactions
A mechanistic examination of salting out in protein–polymer membrane interactions
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DOI：
10.1073/pnas.1909860116
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发表时间：
2019-10
期刊：
影响因子：
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通讯作者：
Nicholas A Moringo;Logan D. C. Bishop;Hao Shen;Anastasiia Misiura;Nicole C. Carrejo;Rashad Baiyasi;
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文献类型：
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作者：
Nicholas A Moringo;Logan D. C. Bishop;Hao Shen;Anastasiia Misiura;Nicole C. Carrejo;Rashad Baiyasi;
基于膜的蛋白质分离被广泛使用，并且越来越多地用于纯化研究和生物制药用蛋白质。与纯化过程中的所有步骤一样，在移动的相中凭经验调节盐浓度以溶解蛋白质混合物的所需组分。关于盐析过程的定量描述不足以进行预测性优化。通过量化的相互作用和动力学的单一蛋白质在膜表面的盐作为盐浓度的增加，我们与机械纳米级的可观察到的改进，在真实的分离中观察到的峰宽。这一结果表明，通过小规模单分子观察得到的模拟可用于优化分离条件，从而实现更有效的分离。开发一个机械的理解蛋白质动力学和构象变化的聚合物界面是一系列的过程，包括工业蛋白质分离的关键。盐析是蛋白质分离中普遍存在的程序的一个例子，但根据经验进行了优化，因为没有允许预测建模的潜在相互作用的机械描述。在这里，我们研究了在盐析条件下使用单分子跟踪和合奏分离相结合的模型转铁蛋白-尼龙系统的峰窄化。不同的表面运输模式和蛋白质的构象变化在带负电荷的尼龙接口被量化为盐浓度的函数。单分子动力学将色谱峰加宽的宏观改善与表面相互作用机制的微观分布（例如连续时间随机游走和简单吸附-解吸）相关联。基于随机色谱理论的Monte Carlo模拟使用从单分子观测中提取的动力学数据进行。模拟结果与实验结果一致，表明随着盐浓度的增加，峰宽减小。结果表明，对膜进行化学修饰，降低表面随机游走的概率，可以减少全规模蛋白质分离中的峰展宽。更广泛地说，这项工作代表了将单分子实验和机械理论相结合的概念证明，以改善昂贵且耗时的经验优化方法。
Significance Membrane-based protein separations are utilized broadly, and increasingly, to purify proteins for research and biopharmaceuticals. Like all steps in the purification process, the salt concentration is adjusted empirically in the mobile phase to elute a desired component of a protein mixture. There is insufficient quantitative description about the salting out process to allow for predictive optimization. By quantifying the interactions and kinetics of single proteins at the surface of a membrane as salt concentration is increased, we relate mechanistic nanoscale observables to an improvement in the peak broadness observed in real separations. This result suggests that simulations, informed by small-scale single-molecule observations, could be used to optimize separation conditions, leading to more efficient separations. Developing a mechanistic understanding of protein dynamics and conformational changes at polymer interfaces is critical for a range of processes including industrial protein separations. Salting out is one example of a procedure that is ubiquitous in protein separations yet is optimized empirically because there is no mechanistic description of the underlying interactions that would allow predictive modeling. Here, we investigate peak narrowing in a model transferrin–nylon system under salting out conditions using a combination of single-molecule tracking and ensemble separations. Distinct surface transport modes and protein conformational changes at the negatively charged nylon interface are quantified as a function of salt concentration. Single-molecule kinetics relate macroscale improvements in chromatographic peak broadening with microscale distributions of surface interaction mechanisms such as continuous-time random walks and simple adsorption–desorption. Monte Carlo simulations underpinned by the stochastic theory of chromatography are performed using kinetic data extracted from single-molecule observations. Simulations agree with experiment, revealing a decrease in peak broadening as the salt concentration increases. The results suggest that chemical modifications to membranes that decrease the probability of surface random walks could reduce peak broadening in full-scale protein separations. More broadly, this work represents a proof of concept for combining single-molecule experiments and a mechanistic theory to improve costly and time-consuming empirical methods of optimization.