Detection and prevention of protein aggregation before, during, and after purification
Detection and prevention of protein aggregation before, during, and after purification
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DOI：
10.1016/s0003-2697(03)00059-9
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发表时间：
2003-05-15
影响因子：
2.9
通讯作者：
Bicknell, A
中科院分区：
文献类型：
--
作者：
Bondos, SE;Bicknell, A
蛋白质用于体外研究或作为治疗剂的使用经常受到表达、纯化、储存或转移到必需的测定缓冲液中期间蛋白质聚集的阻碍。有大量潜在的蛋白质稳定剂可用，但确定哪些是合适的可能需要几天或几周。我们开发了一种溶解度测定法，以确定给定蛋白质的最佳共溶剂，该测定法需要非常少的蛋白质并且仅需几个小时即可完成。该技术通过过滤将天然蛋白与可溶性和不溶性聚集体分离，并通过SDS-PAGE或Western印迹检测两种形式的蛋白。可以同时筛选多种缓冲液以确定增强蛋白质溶解度的条件。混合物和粗裂解物中的单一蛋白质的行为可以用这种技术分析，允许在蛋白质纯化之前和整个蛋白质纯化过程中进行测试。聚集的蛋白质也可以在稳定天然蛋白质的条件下进行测定，然后可以用于改善随后的纯化。该溶解度测定使用大小范围为17至150 kDa的原核和真核蛋白质进行测试，包括单体和多聚体蛋白质。从所呈现的结果来看，这种技术可以应用于各种蛋白质。(C)2003 Elsevier Science（美国）。All rights reserved.
The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins. (C) 2003 Elsevier Science (USA). All rights reserved.