iBiT: A vector system allowing stable genome integration and scalable expression of NanoLuc fusion proteins to monitor dynamic protein-protein interactions in intact cells.

The accurate determination of protein-protein interactions (PPIs) within living cells is essential for our understanding of cellular processes. Molecular interactions can be determined through complementation systems, in which the spatial proximity of two molecular fragments reconstitutes their activity. Among these, NanoLuc Binary Technology (NanoBiT®) utilizes the functional complementation of a large (LgBiT) and a small (SmBiT) luciferase fragment, but its reliability and biological interpretability depends critically on balanced expression of the interacting proteins. To ensure a highly reproducible and finely controlled expression of the interaction partners, we developed a novel plasmid system allowing stable and biallelic integration of NanoLuc plasmids into the safe harbor locus on human chromosome 19. To avoid overexpression artifacts the expression of both interaction partners is precisely regulated by the Tet-On system. We used this inducible binary technology (iBiT) system to investigate a largely unexplored interaction between the full-length isoform 1 and the N-terminally truncated isoform 2 of p62/Sequestosome 1. These experiments demonstrate that cell lines stably expressing iBiT constructs enable highly reproducible, longitudinal, and time-resolved analyses of protein-protein interactions, providing a robust platform for assessing both protein-protein interaction dynamics and their pharmacological modulation in intact cells.