Development of a "Mix and Read" Assay for Target Detection Using Split-Luciferase Reconstitution

Current detection systems used to study protein-protein interactions utilize an elaborate array of chemical or biological labeling of the target for study, and thus have limitations in applications and analysis. To address the shortcomings of the typically used detection systems, such as an ELISA, the goal of this project was to develop a “mix-and-read” assay where a target protein would be detected in a single step, without the need for washes. The system is based on a luciferase complementation assay using binders derived from the Sso7d protein scaffold fused to the components of NanoLuc split-luciferase. Luciferase is a bioluminescent enzyme that is found in many marine organism and arthropods, which scientists have dissected into two non-active components. These components, when brought into close proximity via protein-protein interactions, recombine to form the active luciferase molecule, which catalyzes a light-emitting reaction. For this protein complementation assay, Sso7d-derived binders that recognize a specific target will be fused to the N and C terminus of the split luciferase respectively. When binders are in solution with the target and the two binders bind the target, the luciferase molecule will reconstitute to its active form and with the addition of the reagent, a luminescent signal will be emitted. As a proof of concept, split components of NanoLuciferase, isolated from the deep-sea shrimp Oplophorus gracilirostirs, were fused to two lysozyme Sso7d binders. These preliminary findings show that lysozyme could be detected by this approach even after 3 minutes after the addition of the detection reagent, and the luminescence response increased with lysozyme concentration and incubation time. After validating the system, the next steps involve the development of a systematic approach to isolate Sso7d binders for other targets that could be used with the split luciferase model.