Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby 2800, Denmark.
Joint BioEnergy Institute, Emeryville, California 94608, United States.
ACS Synth Biol. 2021 Mar 19;10(3):466-477. doi: 10.1021/acssynbio.0c00476. Epub 2021 Feb 12.
Protein misfolding and aggregation are linked to neurodegenerative diseases of mammals and suboptimal protein expression within biotechnology. Tools for monitoring protein aggregates are therefore useful for studying disease-related aggregation and for improving soluble protein expression in heterologous hosts for biotechnology purposes. In this work, we developed a promoter-reporter system for aggregated protein on the basis of the yeast native response to misfolded protein. To this end, we first studied the proteome of yeast in response to the expression of folded soluble and aggregation-prone protein baits and identified genes encoding proteins related to protein folding and the response to heat stress as well as the ubiquitin-proteasome system that are over-represented in cells expressing an aggregation-prone protein. From these data, we created and validated promoter-reporter constructs and further engineered the best performing promoters by increasing the copy number of upstream activating sequences and optimization of culture conditions. Our best promoter-reporter has an output dynamic range of approximately 12-fold upon expression of the aggregation-prone protein and responded to increasing levels of aggregated protein. Finally, we demonstrate that the system can discriminate between yeast cells expressing different prion precursor proteins and select the cells expressing folded soluble protein from mixed populations. Our reporter system is thus a simple tool for diagnosing protein aggregates in living cells and should be applicable for the health and biotechnology industries.
蛋白质错误折叠和聚集与哺乳动物的神经退行性疾病以及生物技术中蛋白质表达不佳有关。因此，监测蛋白质聚集的工具对于研究与疾病相关的聚集以及提高生物技术中异源宿主中可溶性蛋白质的表达非常有用。在这项工作中，我们基于酵母对错误折叠蛋白质的天然反应，开发了一种用于聚集蛋白质的启动子报告系统。为此，我们首先研究了酵母在表达折叠可溶性和易于聚集的蛋白质诱饵时的蛋白质组，鉴定了编码与蛋白质折叠和热应激反应相关的蛋白质以及泛素蛋白酶体系统的基因，这些基因在表达易于聚集的蛋白质的细胞中过表达。根据这些数据，我们创建并验证了启动子报告构建体，并通过增加上游激活序列的拷贝数和优化培养条件进一步对最佳性能的启动子进行了工程改造。当表达易于聚集的蛋白质时，我们最好的启动子报告具有约 12 倍的输出动态范围，并对聚集的蛋白质水平增加做出反应。最后，我们证明该系统可以区分表达不同朊病毒前体蛋白的酵母细胞，并从混合群体中选择表达折叠可溶性蛋白的细胞。因此，我们的报告系统是一种用于诊断活细胞中蛋白质聚集的简单工具，应该适用于健康和生物技术行业。
