Churion Kelly A, Bondos Sarah E
Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX, USA.
Methods Mol Biol. 2012;896:415-27. doi: 10.1007/978-1-4614-3704-8_28.
Intrinsically disordered proteins are anticipated to be more prone to aggregation than folded, stable proteins. Chemical additives included in the buffer can help maintain proteins in a soluble, monomeric state. However, the array of chemicals that impact protein solubility is staggering, precluding iterative testing of chemical conditions during purification. Herein, we describe a filter-based aggregation assay to rapidly identify chemical additives that maintain solubility for a protein of interest. A hierarchical approach to buffer selection is provided, in which the type of chemical which best improves solubility is first determined, followed by identifying the optimal chemical and its most effective concentration. Finally, combinations of chemical additives can be assessed if necessary. Although this assay can be applied to purified protein, partially purified protein, or aggregated protein, this protocol specifically details the use of this assay for crude cell lysate. This approach allows identification of solubility-promoting buffers prior to the initial protein purification.
预计内在无序蛋白比折叠的稳定蛋白更容易聚集。缓冲液中包含的化学添加剂有助于将蛋白质维持在可溶的单体状态。然而，影响蛋白质溶解度的化学物质种类繁多，排除了在纯化过程中对化学条件进行反复测试的可能性。在此，我们描述了一种基于过滤的聚集分析方法，以快速鉴定能维持目标蛋白溶解度的化学添加剂。提供了一种分级缓冲液选择方法，首先确定最能提高溶解度的化学物质类型，然后确定最佳化学物质及其最有效浓度。最后，如有必要，可以评估化学添加剂的组合。尽管该分析方法可应用于纯化蛋白、部分纯化蛋白或聚集蛋白，但本方案具体详细说明了该分析方法用于粗细胞裂解物的使用方法。这种方法允许在初始蛋白质纯化之前鉴定促进溶解度的缓冲液。
