Luminescence complementation assay for measurement of binding to protein C-termini in live cells.

ABSTRACT: Protein-protein interactions (PPIs) involving the extreme C-terminus serve important scaffolding and regulatory functions. Here, we leveraged NanoBiT technology to build a luminescent complementation assay for use in studying this subcategory of PPI. As a model system, we fused one component of NanoBiT to the disordered C-terminus of heat shock protein (Hsp70) and the other to its binding partner, the tetratricopeptide repeat (TPR) domain of CHIP/STUB1. We found that HEK293 cells that stably express these chimeras under a doxycycline promoter produced a robust luminescence signal. This signal was sensitive to mutations and it was further tuned by the expression of competitive C-termini. Using this system, we identified a promising, membrane permeable inhibitor of the Hsp70-CHIP interaction

SUBMITTER: Nadel CM 

PROVIDER: S-EPMC8054679 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature