Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor.
Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor.
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DOI：
10.3791/62628
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发表时间：
2021-06-29
影响因子：
1.2
通讯作者：
Dahl, Jan-Ulrik
中科院分区：
文献类型：
--
作者：
Sultana, Sadia;Anderson, Greg M.;Hoffmann, Kevin Pierre;Dahl, Jan-Ulrik
活生物体暴露于环境和细胞应激通常会导致蛋白质稳态的破坏，并可能导致蛋白质聚集。蛋白质聚集体在细菌细胞中的积累可导致细胞表型行为的显著改变，包括生长速率、胁迫抗性和毒力的降低。存在几种实验程序用于检查这些应激物介导的表型。本文描述了一种优化的检测方法，用于提取和可视化不同大肠杆菌菌株用含银钌抗菌剂处理后的聚集和可溶性蛋白质。已知该化合物产生活性氧并引起广泛的蛋白质聚集。该方法结合了基于离心的蛋白质聚集体和可溶性蛋白质从处理和未处理的细胞与随后的分离和可视化的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和考马斯染色。该方法简单、快速，并可定性比较不同大肠杆菌中蛋白聚集体的形成。大肠杆菌菌株。该方法具有广泛的应用范围，包括研究其他蛋白毒性抗菌剂对多种细菌体内蛋白质聚集的影响的可能性。此外，该方案可用于鉴定有助于增加对蛋白毒性物质的抗性的基因。凝胶带可用于随后鉴定特别易于聚集的蛋白质。
The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.
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影响因子：
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作者：
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通讯作者：
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