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bioRxiv · 10.1101/2023.08.31.555680

Improving split reporters of protein-protein interactions through orthology-based protein engineering

Rakotoarison, L.-M.·Tebo, A. G.·Böken, D.·Gautier, A.
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Abstract

Protein-protein interactions (PPI) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of RspA-splitFAST, an improved reporter displaying higher brightness, lower self-complementation and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, RspA-splitFAST is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

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Rakotoarison, L.-M., Tebo, A. G., Böken, D., Gautier, A.. 2023-09-01. Improving split reporters of protein-protein interactions through orthology-based protein engineering. https://doi.org/10.1101/2023.08.31.555680

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