Construction of protein-loadable protein cages using the hybrid proteins of the oleosin hydrophobic domain and hydrophilic dimeric coiled-coil

Abstract We successfully designed the bilayer-capsule-formable OLE-ZIP proteins by fusing the dimeric coiled coil motif (NZ and CZ) [1] and the hydrophobic domain OLE(56–106) of the sunflower-derived oleosin. In order to improve handling in a buffer, we characterized the fusion protein with thioredoxin, Trx-OLE-ZIP as an alternative. Based on the emulsion method, Trx-OLE-ZIP could successfully form stable liposome-kind bilayer capsules, with empty hollow. Furthermore, it could encapsulate other water-soluble proteins, such as GFP, to the internal hollow with a buffer of inner aqueous phase. By mutating four conserved Thr residues (Thr59, Thr66, Thr97, and Thr104) in the OLE(56–106) domain with Asp residues, we further successfully constructed the pH-sensitive mutant, Trx-OLE-D-ZIP. Under weakly acidic conditions at pH = 5, Trx-OLE-D-ZIP could maintain a spherical cage-morphology. By shifting the solution pH to basic (pH = 9), the protein-cages spontaneously collapsed and released the loaded proteins.