Internally twin-Strep tagged CD63 for rapid and efficient isolation of engineered extracellular vesicles encapsulating functional proteins

Extracellular vesicles (EVs) are important mediators of intercellular communication. Achieving high purity of intact engineered EVs through separation from the overwhelming background of unmodified vesicles and cellular or serum-derived contaminants remains a major challenge, yet is critical for fundamental studies of EV biology and EV-based therapeutics. To address this unmet need, we applied structure-guided engineering to canonical EV tetraspanins. Focusing on CD63 and CD9, we identified sites for internal insertion of the twin-Strep tag via flexible linkers into unstructured regions of their large extracellular loops. This design retains functional properties of the scaffolds while providing a robust molecular handle enabling rapid, efficient, and selective antibody-free affinity-based isolation of intact EVs using advanced Strep technology. Fusing proteins of interest (POIs) to the C-terminus of these scaffolds, either directly or via a photoactivatable protein, allowed POI loading into the lumen of twin-Strep tagged EVs with optional optogenetic control of POI release. Proteomic analysis confirmed the high purity of captured engineered EVs with >96% of contaminants removed, enabling sensitive detection of numerous EV-associated proteins. We also report the efficient removal of contaminating virus particles resembling EVs in size and density. Purified engineered EVs successfully delivered an encapsulated fluorescent reporter into target cells, bypassing dye labeling commonly used to track EVs. We demonstrate endosomal escape of the reporter, facilitated by EV decoration with vesicular stomatitis virus glycoprotein, and cytoplasmic release upon optogenetic activation. Our toolbox may serve as a broadly applicable strategy for the efficient production of intact and highly pure engineered EVs to support potential fundamental or translational EV studies.