Engineering Magnetic Beads for Affinity Enrichment of Exosomes.

Exosomes play a crucial role in intercellular communication and disease diagnostics, yet their affinity enrichment remains challenging due to the heterogeneous expression of surface markers. To address this, we engineered a recombinant fusion protein, ExoBp, designed to bind multiple exosome surface biomarkers through integrated peptide motifs and protein domains with sterically unhindered conformation revealed by structure prediction. The constructed protein was expressed but required denaturing conditions for purification. Despite refolding challenges, ExoBp was successfully immobilized onto streptavidin-coated magnetic beads in its denatured state, followed by on-bead refolding to restore functionality. The resulting ExoBp magnetic beads demonstrated efficient exosome binding, as confirmed by the detection of exosomal markers after coincubation with exosome derived from different types of cells. Both dialysis- and dilution-based refolding methods yielded stable ExoBp-bead complexes with retained binding capacity. This study presents a novel, affinity-based exosome enrichment tool leveraging multitarget binding, offering a promising platform for exosome isolation and downstream biomedical applications.