Translation-coupled protein folding assay using a protease to monitor the folding status
Translation-coupled protein folding assay using a protease to monitor the folding status
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DOI：
10.1002/pro.3624
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发表时间：
2019-07-01
期刊：
影响因子：
8
通讯作者：
Taguchi, Hideki
中科院分区：
文献类型：
--
作者：
Niwa, Tatsuya;Uemura, Eri;Taguchi, Hideki
蛋白质折叠是蛋白质执行几乎所有细胞功能的必要先决条件。人们越来越需要一种简单而可靠的方法来研究相同条件下大规模的蛋白质折叠。我们之前开发了一种全球折叠分析系统，其中使用大肠杆菌为基础的无细胞翻译系统翻译的蛋白质被离心以定量上清部分。虽然分析是基于假设上清含有折叠的天然状态，但上清也包括非天然的非结构蛋白质。一般来说，蛋白酶可以识别和降解非结构化蛋白质，因此我们使用蛋白酶来消化非结构化区域以监测折叠状态。在蛋白质翻译过程中添加Lon蛋白酶，不仅在可溶性部分，而且在易于聚集的部分，揭示了隐藏的亚组分。我们在蛋白酶包涵试验中翻译了90种大肠杆菌蛋白，无论是否存在伴侣蛋白。折叠试验，它揭示了分子机制下的聚集形成和伴侣效应，可以应用于大规模的分析。
Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large-scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated similar to 90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.