A Novel Technology Platform for Extracellular Vesicle-Targeted Expression of Drug-Metabolizing Enzymes: Driving...

Background: Cytochrome P450 3A4 (CYP3A4) is a key membrane-anchored drug-metabolizing enzyme. Its expression and purification in heterologous systems are severely hindered by low yield and detergent-induced structural inactivation. Although extracellular vesicles (EVs) provide an ideal natural lipid bilayer environment to stabilize membrane proteins, targeted loading remains challenging. The ESCRT and ALIX-binding region (EABR) of CEP55 can efficiently recruit core components of the endosomal sorting complex (ESCRT) to mediate membrane fission. Objectives: This study used the EABR motif to drive the targeted vesicular secretion of CYP3A4, thereby establishing a novel membrane protein engineering platform. Methods and Results: EABR was fused with fluorescent protein, confirming its specific mediation of vesicular secretion. Recombinant plasmids of EABR/CYP3A4 and its reverse mutant (R-EABR) were transfected into HEK293T cells. Western blot and midazolam-based metabolic assays showed that forward EABR significantly enhanced CYP3A4 expression and EV secretion, while R-EABR lost exocytosis function. EVs isolated by ultracentrifugation verified EABR's role in recruiting ESCRT and improving CYP3A4 activity. Conclusions: Forward CEP55-EABR specifically and efficiently drives vesicular encapsulation of CYP3A4, enhancing its expression and secretion. This ESCRT-mediated strategy avoids destructive purification, provides a stable lipid-rich bioreactor for CYP3A4, and has great translational potential in high-throughput in vitro drug metabolism and screening platforms.