Preparation of Membrane Protein&#8211;Functionalized Polymer and Polymer/Lipid Hybrid Large and Giant Unilamellar Vesicles

Nika Otrin; Chee Seng Man; Wenqi Huang; Kai Sundmacher; Lado Otrin

doi:10.3791/71699

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Method Article

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

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DOI:

10.3791/71699

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August 7th, 2026

Nika Otrin¹ , Chee Seng Man¹^,² , Wenqi Huang¹ , Kai Sundmacher¹^,²^,³ , Lado Otrin¹

¹Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, ²Max Planck School Matter to Life, ³Process Systems Engineering, Otto von Guericke University

Corresponding Authors: Nika Otrin <nika.otrin@mpi-magdeburg.mpg.de>

Summary

This protocol demonstrates detergent-mediated reconstitution of a membrane protein, cytochrome bo3 ubiquinol oxidase, into large unilamellar vesicles made of PDMS-g-PEO and their conversion into giant vesicles via fusion–electroformation. The resulting functional polymer-based compartments are suitable for advanced biochemical and biophysical studies.

Abstract

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.

Introduction

Amphiphilic block copolymers can self-assemble in aqueous solution into membranes, which spontaneously curve and close upon themselves to form spherical, membrane-enclosed compartments known as polymersomes (polymer vesicles). Compared to lipid membranes, polymer-based compartments offer increased chemical versatility and enhanced chemical¹ and mechanical stability², making them highly attractive for applications such as drug delivery, nano- and microreactors, and the construction of durable artificial cells. Both our group and others have functionalized polymer membranes by inserting membrane proteins to create artifici....