Production and characterization of escherichia coli outer membrane vesicles displaying anti-muc1 single-chain variable fragment via spytag/spycatcher system

Abstract

Outer membrane vesicles (OMVs) secreted from Gram-negative bacteria have been discovered for a decade. Special features like high stability, ease of production, and intrinsic adjuvanticity highlight the use of OMVs in diverse applications such as vaccination, bioimaging, and multifunctional delivery systems. Synthetic biological approaches can functionalize OMVs to present heterologous proteins by expressing genetic fusion of target protein to outer membrane proteins. However, complex proteins cannot be incorporated into the OMVs due to lack of some post-translational modifications in bacteria. Herein, we demonstrated a convenient method for presentation of versatile proteins in OMVs with the implementation of the bio-ligation system called SpyTag/SpyCatcher system. OMVs derived from Escherichia coli were generated to anchor SpyCatcher via Lpp’OmpA surface display system (OMVs:Lpp’OmpA-SpyCatcher). To test the proof-of-concept, anti-MUC1 single-chain variable fragment (scFv) clone SM3 which previously used in targeting MUC1-presenting tumor cells was chosen and produced as a fusion with SpyTag (SpyTag-SM3) in CHO-based expression system. This system provides disulfide linkage processes to maintain its function. Upon mixing, SpyTag simultaneously performs an irreversible isopeptide bond with SpyCatcher resulting in coupling of SpyTag-SM3 onto the surface of OMVs:Lpp’OmpA-SpyCatcher. The conjugation of two proteins on OMVs was confirmed by Western blotting. Physiochemical characterizations of conjugated OMVs were also analyzed by Dynamic light scattering and Transmission electron microscope. Despite attachment of anti-MUC1 scFv SM3, the morphology of conjugated OMVs was spherical lipid vesicles with the size of 103.77 nm. In addition, the localization of scFv SM3 was observed on the surface of the OMVs as evaluated by proteinase K protection assay. Furthermore, the result of binding analysis towards MUC1-presenting cell (MCF-7) indicated the binding of anti-MUC1 scFv SM3 displayed on the conjugated OMVs. As a result, this study provides a flexible protein decoration system in OMVs that can be used in targeted drug delivery system.