Preparation of Membrane Protein-Functionalized Polymer and Polymer/Lipid Hybrid Large and Giant Unilamellar Vesicles.

Vesicles formed from amphiphilic copolymers, alone or blended with phospholipids, offer superior mechanical and chemical stability compared to conventional lipid vesicles. This makes them an attractive chassis, one that can be further developed and expanded to enable specific applications, such as drug delivery, diagnostic biosensing, construction of artificial cells, and bioinspired micro- and nanoreactors. In particular, functionalization through membrane protein incorporation is essential for many of these applications. Here, we present a protocol for preparing membrane protein-functionalized large unilamellar vesicles (LUVs) from the graft copolymer PDMS-g-PEO via detergent-mediated reconstitution. Furthermore, we describe how these large vesicles can be converted to giant unilamellar vesicles (GUVs) using a fusion-electroformation approach. The protocol covers fluorescence labeling of membrane proteins, preparation of polymer and hybrid LUVs, membrane protein reconstitution, size distribution analysis via dynamic light scattering (DLS), assessment of protein activity via oxygen consumption measurements, preparation of protein-functionalized GUVs, and analysis of protein insertion and proton pumping activity in GUVs via confocal microscopy. Representative results demonstrate the formation of monodisperse proteo-LUVs with a polydispersity index (PDI) below 0.2, and successful generation of proteo-GUVs ranging from 5-35 µm in diameter. Protein activity is confirmed by oxygen consumption measurements in both polymer LUVs (18.2 nmol/min/mL) and hybrid LUVs (26.9 nmol/min/mL). Protein insertion into GUVs is quantified via fluorescence intensity, yielding 20.6 ± 3.7 a.u. for polymer GUVs and 26.2 ± 5.0 a.u. for hybrid GUVs. Proton pumping activity in GUVs, monitored via an encapsulated pH-sensitive dye, is consistent with protein functionality, with inward proton pumping being predominant. The copolymer's mechanical softness and lipid-like bilayer thickness (~5.3 nm) support efficient protein insertion and preservation of functionality.