Engineering GFP–NanoLuc fusion substrates for sensitive, quantitative detection of protease activity via BRET

Proteases play essential roles in diverse biological processes and are closely associated with various diseases, making them important targets for diagnostics and therapeutic development. Conventional protease activity assays often rely on chromogenic or fluorogenic substrates that require organic synthesis, increasing production costs and limiting accessibility. To address these, we developed a genetically encoded, bioluminescence resonance energy transfer (BRET)-based protease substrate composed of NanoLuc (NLuc) and green fluorescent protein (GFP) fused to either end of a cleavable peptide sequence. In the intact fusion protein, NLuc luminescence excites GFP via BRET, resulting in green emission. Proteolytic cleavage disrupts BRET, reducing GFP fluorescence. Using tobacco etch virus (TEV) protease as a model, we demonstrated that introducing glycine-serine linkers flanking the cleavage site (TEVcs) enhances proteolytic accessibility and signal responsiveness. The optimized substrate enabled quantitative detection of TEV protease activity with a detection limit of 0.0426 μM. Furthermore, substituting the TEVcs with a caspase-3-specific sequence allowed sensitive detection of caspase-3, with a limit of 0.62 nM. This system offers a cost-effective and broadly applicable platform for real-time protease activity measurement using standard Escherichia coli expression systems, eliminating the need for chemically synthesized substrates.