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Get Free AccessProtein 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.
David Romero-Suárez, Tune Wulff, Yixin Rong, Tadas Jakočiūnas, Satoshi Yuzawa, Jay D Keasling, Michael K. Jensen (2021). A Reporter System for Cytosolic Protein Aggregates in Yeast. , 10(3), DOI: https://doi.org/10.1021/acssynbio.0c00476.
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Type
Article
Year
2021
Authors
7
Datasets
0
Total Files
0
Language
en
DOI
https://doi.org/10.1021/acssynbio.0c00476
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