This article discusses the use of a nanoluciferase-based reporter system for detecting the receptor binding domain of the SARS-CoV-2 spike glycoprotein. The system utilizes a split version of luciferase, allowing for the reconstitution of a functional enzyme that emits a luminescent signal proportional to the target protein concentration.
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.
To evaluate the luminescent signal from the bioreporter using a luciferase assay, first, prepare the reaction components, and use the supernatant as the source of the bioreporter.
Dilute the LgBiT and substrate to 1x before use. Transfer 50 microliters of the supernatant from each well or tube, to a 96-well plate, and add 50 microliters of 1x LgBiT to each well. Incubate for 5 minutes at room temperature.
Open the luminometer software. Add 50 microliters of 1x substrate to each well. Place the plate in the luminometer and run the software.
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