Anchoring Giant Plasma Membrane Vesicles to a Surface for Novel Biosensing

Abstract

Giant plasma membrane vesicles (GPMVs) are unilamellar bodies that can be easily isolated from mammalian cells. SNAP-tag is a protein tag which binds covalently with benzylguanine (BG) derivatives, and is also capable of being fused to a protein of interest. Currently, specifically anchoring cellular material remains a challenge for bioengineering. The purpose of our project was to create a BG-functionalized surface to immobilize GPMVs expressing SNAP-tag fused to A2A, a G-protein coupled receptor. We harvested GPMVs from wildtype HEK cells transfected with pSNAPf-A2A DNA through addition of PFA/DTT solution. The surface was constructed through a series of thiol-ene click reactions and functionalized with benzylguanine. Live-cell imaging of SNAP-A2A-expressing GPMVs interacting with the surface revealed strong immobilization compared to cells transfected with only A2A and SNAP-A2A-expressing GPMVs on a PEG-thiol functionalized surface. Horizontally-oriented storage of GPMVs immobilized on a BG-functionalized surface maintained the integrity of vesicles and ensured viability after two days. Modification of the surface also produced a gradient of benzylguanine functionalization that selectively immobilized GPMVs, though the exact interaction between BG and SNAP-tag should be further characterized. This novel technique to anchor vesicles from mammalian cells could be instrumental for the development of functional biosensors. Using this anchoring system, a wide variety of compounds could be quickly detected via binding to an exogenously expressed protein of interest within the same membrane environment.