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  5. A Reporter System for Cytosolic Protein Aggregates in Yeast

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Article
en
2021

A Reporter System for Cytosolic Protein Aggregates in Yeast

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0 Files

en
2021
Vol 10 (3)
Vol. 10
DOI: 10.1021/acssynbio.0c00476

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Jay D Keasling
Jay D Keasling

University of California, Berkeley

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David Romero-Suárez
Tune Wulff
Yixin Rong
+4 more

Abstract

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.

How to cite this publication

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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Publication Details

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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