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bioRxiv · 10.1101/580845

Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

Kim, C. K.·Cho, K. F.·Kim, M. W.·Ting, A. Y.
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Abstract

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested \"AND\" gate design of SPARK2--in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest--yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

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Kim, C. K., Cho, K. F., Kim, M. W., Ting, A. Y.. 2019-03-18. Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions. https://doi.org/10.1101/580845

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