Splitluciferase

splitluciferase is a molecular biology tool used to study protein–protein interactions, enzyme activity, and signal transduction in living cells and organisms. The method is based on the concept of split-luciferase complementation, where the luciferase enzyme, normally catalyzing the oxidation of luciferin to produce bioluminescence, is divided into two inactive fragments. Each fragment is genetically fused to a protein of interest. When the two proteins interact, the luciferase fragments are brought into close proximity and reassemble into an active enzyme, resulting in a measurable light signal proportional to the interaction strength.

The approach was first developed with firefly luciferase (photinus pyralis) and has since been adapted to other luciferases such as nanoLuc from the African chelid beetle and Renilla luciferase. Splitluciferase assays are advantageous because they are non‑invasive, allow real‑time monitoring, and have high signal-to-noise ratios. Compared to other reporter systems, they do not require cell fixation or permeabilization and can be performed in vivo.

Applications include screening of drug candidates that disrupt or promote protein interactions, monitoring of enzyme activation, and spatial-temporal studies of signaling pathways. Variants such as NanoBiT use two subunits of nanoLuc engineered for improved foldability and low background. Limitations include potential steric hindrance from fusion tags, possible incomplete complementation, and reliance on appropriate expression levels. Despite these challenges, splitluciferase remains a widely adopted, versatile assay in biochemical and cell‑biological research.