Research from the Skretas Lab Optimizes a High-Throughput Screen for Monitoring Disease-Associated Protein Misfolding and Aggregation in Bacteria.
Skretas Lab has been using the fluorescence of Escherichia coli bacteria that produce fusions of misfolding-prone (MisP) human proteins with green fluorescent protein (GFP) as a high-throughput screen readout for identifying chemical rescuers of disease-related protein misfolding and aggregation.
In a new study recently published in ACS Synthetic Biology, the team identified conditions that lead to optimized performance in this assay. Using misfolding-prone p53 variants associated with cancer as a test case, they showed that strong overexpression conditions, such as high copy number vectors, strong promoters, high inducer concentrations, and high overexpression temperatures, can yield an enhanced dynamic range and sensitivity for the assay. These assay conditions were also found to be optimal for additional MisPs, such as amyloid-β peptide (Aβ) and variants of Cu/Zn superoxide dismutase, associated with Alzheimer’s disease and amyotrophic lateral sclerosis, respectively. At the same time, the team observed that certain conditions, such as inducer concentrations and overexpression temperature, may need to be precisely fine-tuned for each new MisP target to yield optimal assay performance. These findings provide a framework for standardizing MisP-GFP screening assays, thus facilitating their broad application in the discovery of therapeutic agents targeting protein misfolding and aggregation.
Delivoria, D. C., Konia, E., Matis, I., & Skretas, G. (2025). Optimization of a High-Throughput Screen for Monitoring Disease-Associated Protein Misfolding and Aggregation in Bacteria. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.5c00166