Development of a Bioluminescence Assay for Bir2- Caspase3 Interaction Through Split Luciferase Complementary Assay Publisher



Mostafavi M¹ ; Ataei F¹ ; Hamidieh AA² ; Hosseinkhani S¹ Show Affiliations
Source: Biochemical Engineering Journal Published:2022

Abstract

Harnessing of firefly luciferase biochemistry enabled to develop bioluminescent assay for different intracellular protein-protein interactions. Caspase-3 and XIAP are two important players in maintaining the balance of cell death and survival. Linker-BIR2 from XIAP is known to be sufficient for inhibition of caspase-3. Here, caspase-3 and Linker-BIR2 were used for designing two reporters based on split luciferase complementary assay to develop a luminescent probe. Caspase3-CLuc and BIR2-NLuc fragments were ligated in pET-28a (+), overexpressed in E. coli BL21 (DE3) and purified to homogeneity. Interaction between caspase-3 and BIR2 was evaluated by different experimental methods such as luciferase and caspase-3 activity, directed mutagenesis and ELISA, which approved their specific interaction. Next, the developed reporters were employed for analyzing four compounds (Sulphadimidin, Glyburide, Carboplatin and Cetirizine) that showed effects on caspase-3 and BIR2 interaction with the largest effect for Carboplatin. Molecular docking was also showed the largest binding affinity for Glyburide. In conclusion, the developed assay showed specific interaction with light generation, and ability to analyze some chemicals in vitro. © 2022 Elsevier B.V.