Establishment of a BRET-based fusion protein expression system to detect benzo[a]pyrene among polycyclic aromatic...

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants associated with various health risks. They primarily enter the human body through food. The most toxic PAH is benzo[a]pyrene (BaP), which serves as a key marker in food safety regulations. In this study, a NanoBRET-based detection system was developed to evaluate the protein-protein interaction between aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) in response to BaP. BRET vectors encoding NanoLuc-fused AhR and HaloTag-fused ARNT were used to monitor heterodimer formation, and the optimal combination for BaP detection was selected. A cell-free assay was established to assess the biological activity of BaP by quantifying AhR/ARNT heterodimerization. This assay could be used for faster, simpler, and more biologically relevant toxicity evaluation than conventional methods. The assay was optimized to minimize protein consumption and enable high-throughput screening. Thus, this BRET-based platform provides a rapid and accurate method for detecting BaP in food safety applications.