The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters

Luciferase is an enzyme that oxidizes its substrate to form a bioluminescent product.

This property of luciferase is helpful for designing a nanoluciferase-based reporter system in which the split version of the enzyme is used. In this system, the smaller and larger fragments possess a high binding affinity towards each other and can reconstitute the functional enzyme.

To use the nanoluciferase-based system for detecting the target protein - receptor binding domain of SARS-CoV-2 spike glycoprotein - begin with a culture dish containing adherent cells. These cells contain a plasmid encoding the target protein in conjunction with a sequence-specific for the smaller fragment of engineered nanoluciferase.

Upon expression, the cultured cells produce bioreporters - recombinant target proteins fused with luciferase's smaller fragment. Treat the cells with a lysis buffer, and incubate under agitation. The buffer components rapidly lyse the cells - releasing the cellular content, including the bioreporters, into the supernatant.

Transfer the supernatant to a multiwell assay plate. Supplement each well with the solution containing the large fragments of split nanoluciferase, and incubate.

During incubation, the large fragment spontaneously binds with the small subunit of the bioreporter and attains a functional conformation. Add a solution containing furimazine - the substrate, and place the assay plate in a luminometer.

The functional enzyme oxidizes furimazine, emitting a bright luminescent signal. The magnitude of the signal correlates with the target protein concentration.