Name: Preparation of Membrane Protein–Functionalized Polymer and Polymer/Lipid Hybrid Large and Giant Unilamellar Vesicles (Video) | JoVE Journal
Uploaded: 2026-08-07T15:55:00.000+00:00
Duration: 9 min 39 s
Description: Learn to prepare membrane protein-functionalized polymer and hybrid vesicles using detergent-mediated reconstitution. This protocol enables stable LUV and GUV formation for drug delivery, biosensing, and artificial cell applications.

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This research was conducted within the Max Planck School Matter to Life, supported by the Dieter Schwarz Foundation in collaboration with the Max Planck Society. We are grateful to Claudia Bednarz for the isolation and purification of cytochrome bo3 oxidase, and to Anne Christin Weinrich for her assistance with vesicle preparation and microscopy.

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The authors declare no competing financial interests.

Polymersomes and polymer/lipid hybrid vesicles exhibit enhanced chemical and mechanical stability compared to conventional lipid vesicles, making them highly attractive for applications ranging from drug delivery and biosensing to nano- and microreactors and artificial cells. However, their distinct membrane architecture and biophysical properties often prevent the direct transfer of protocols optimized for lipid vesicles to polymer-based systems15,20,...

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 artificial organelles and cells1,3,4,5,6,7,8,9,10,11,12,13,14,15. Polymers containing a poly(dimethylsiloxane) (PDMS) hydrophobic core are particularly well suited for membrane protein incorporation due to the high chain flexibility and the possibility to tune the membrane hydrophobic thickness to better match the protein&#8217;s transmembrane domain5. While our laboratory has primarily concentrated on the graft copolymer poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO), which forms highly dynamic membranes4,16,17 that are softer than conventional lipid bilayers and exhibit a thickness of approximately 5.3 nm1, other groups have successfully employed PDMS-based triblock copolymers such as PMOXA-b-PDMS-b-PMOXA5,6,7,8,9,10,11 and PEtOz-b-PDMS-b-PEtOz12 for membrane protein reconstitution. Even comparatively rigid diblock copolymers such as PBD-b-PEO, particularly in hybrid systems mixed with lipids, have been utilized for successful protein incorporation13. These examples highlight the versatility of polymer-based membranes as reconstitution platforms, which can additionally provide enhanced chemical stability1 and prolonged functional lifetime14 for incorporated membrane proteins. Although polymer/lipid hybrid membranes may not always match the chemical stability of pure polymer systems1, they offer an attractive compromise by combining the mechanical robustness of polymers with the biological relevance of lipids. This results in a membrane environment that more closely mimics native biological membranes while preserving key advantages of synthetic materials. Such hybrid architectures can offer additional advantages, including reduced proton permeability arising from nanoscale rearrangement1 and domain formation within the membrane15.
Several techniques exist for reconstituting membrane proteins into nanosized vesicles (LUVs), including organic solvent-mediated reconstitution (e.g., reverse-phase evaporation, rehydration of lipid-protein films), mechanical approaches (e.g., sonication, French press, freeze-thaw), and detergent-mediated reconstitution (via detergent removal, dilution, or direct incorporation)18, and more recent strategies such as direct transfer from styrene-maleic acid copolymer nanodiscs19. While porins have been successfully incorporated into polymer LUVs through direct insertion, for example, by adding the protein to the polymer film during rehydration8. This approach generally results in low insertion efficiency for larger or more complex membrane proteins and often leads to random protein orientation. Consequently, detergent-mediated reconstitution, which involves controlled addition of detergent followed by its removal, remains the most widely used method. It is also the preferred strategy for incorporating larger membrane proteins into polymer-based LUVs, particularly for asymmetric proteins where controlled orientation is crucial for function. In this approach, membranes of vesicles are first saturated with the detergent of choice (saturating detergent concentration is denoted as Rsat), partially solubilized (coexistence of vesicles and micelles), or fully solubilized, i.e., broken down into micelles (detergent concentration is denoted as Rsol). This initial membrane treatment is then followed by protein addition, incubation, and detergent removal18. The latter can be achieved via dialysis, size-exclusion chromatography, or adsorption onto hydrophobic polystyrene beads (e.g., Bio-Beads). The choice of detergent, its concentration, and the removal method are optimized for each membrane type and protein to maximize incorporation efficiency and control protein orientation20.
While LUVs are well-suited for bulk activity measurements, single-vesicle studies and optical microscopy&#8211;based analyses require membrane proteins to be reconstituted into giant unilamellar vesicles (GUVs). Five main approaches are used for protein incorporation into GUVs: co-formation in the presence of protein and organic solvent21, co-formation in the presence of a detergent22, insertion into preformed GUVs via detergent dilution23, insertion via membrane fusion with proteo-LUVs16,24, and fusion-electroformation25. While these approaches have been successfully applied to lipid GUVs, their transfer to polymer or polymer/lipid hybrid GUVs is often challenging. In particular, co-formation and detergent-dilution methods frequently result in low insertion efficiencies in polymer membranes or are incompatible with certain polymer compositions15. Fusion-based strategies4,16,24,26,27 that rely on additional fusogenic agents may also be problematic, as synthetic cationic lipids/polymers or fusogenic peptides can compromise membrane protein stability, alter membrane properties, or reduce the incorporation efficiency of the target protein. Fusion-electroformation overcomes many of these limitations and has proven to be a robust and efficient method for incorporating membrane proteins into polymer and polymer/lipid hybrid GUVs1,5,15. This approach enables controlled protein incorporation while preserving membrane integrity and protein functionality, making it particularly suitable for the generation of protein-functionalized polymer GUVs.
Reconstituting proton pumps into polymer-based vesicles is essential for constructing durable artificial cells, as it enables the generation of transmembrane proton gradients&#8212;the fundamental energy currency of living systems. By recreating chemiosmotic energy conversion in a controlled minimal membrane environment28, these vesicles can drive ATP synthesis, active transport, and metabolic reactions, establishing the energetic foundation required for autonomous, life-like functionality. One representative proton pump used in such systems is cytochrome bo3 oxidase from E. coli, a terminal respiratory enzyme that couples oxygen reduction to proton translocation across the membrane. Because electron transfer and proton pumping are tightly coupled, their activity in synthetic vesicles can be quantitatively monitored either by oxygen consumption1,20 or by tracking proton gradient formation1.
In this protocol, we present a detailed workflow for the preparation of PDMS-g-PEO-based LUVs, including pure polymer and polymer/lipid hybrid membranes, their functionalization with membrane proteins, scale-up to GUVs, and activity measurements of the reconstituted proton pump cytochrome bo3 oxidase through both bulk oxygen consumption assays and single-vesicle proton pumping measurements.

<table><tbody><tr><td>&amp;Auml;KTA pure&amp;trade; chromatography system</td><td>Cytiva</td><td>29018224</td><td></td></tr><tr><td>18:1 Liss Rhod PE</td><td>Avanti Research</td><td>810150</td><td></td></tr><tr><td>8 well chambered coverglass</td><td>Cellvis</td><td>C8-1.5H-N</td><td></td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters</td><td>Sigma Aldrich</td><td>UFC5030</td><td></td></tr><tr><td>ATTO 643 NHS ester</td><td>ATTO-TEC</td><td>AD 643-31</td><td></td></tr><tr><td>Bio-Beads SM-2</td><td>Bio-Rad</td><td>1523920</td><td></td></tr><tr><td>Cholic acid-Na-salt (sodium cholate)</td><td>Serva</td><td>361-09-1</td><td></td></tr><tr><td>Coenzyme Q1</td><td>TargetMol</td><td>TGM-T19594</td><td></td></tr><tr><td>Confocal microscope STELLARIS 5</td><td>Leica Microsystems</td><td>603810</td><td></td></tr><tr><td>DTT (Dithiothreitol)</td><td>Merck</td><td>10197777001</td><td></td></tr><tr><td>Fine Tip Ultra Micro Pastette</td><td>Pastette</td><td>LW4243</td><td></td></tr><tr><td>HPTS (pyranine, 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt)&amp;nbsp;</td><td>Sigma Aldrich</td><td>6358-69-6</td><td></td></tr><tr><td>ITO-coated glass slides (25&amp;times;75&amp;times;1.1 mm, &amp;le;20 Ohm/sq.)</td><td>pgo</td><td>CEC020S</td><td></td></tr><tr><td>LAS X software</td><td>Leica</td><td>https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/</td><td></td></tr><tr><td>Mini centrifuge MCF-2360</td><td>LMS</td><td>24572</td><td></td></tr><tr><td>Mini-extruder</td><td>Avanti Research</td><td>610000</td><td></td></tr><tr><td>Oxytherm+P system</td><td>Hansatech Instruments</td><td>/</td><td></td></tr><tr><td>Plasma cleaner (benchtop)</td><td>Harrick Plasma</td><td>PDC-32G-2</td><td></td></tr><tr><td>Polycarbonate membranes 0.1 &amp;mu;m</td><td>Avanti Research</td><td>610005</td><td></td></tr><tr><td>Press-to-seal silicone isolator (round, 20 mm diameter, 1 mm depth)</td><td>Grace Bio-Labs&amp;nbsp;</td><td>41161502</td><td></td></tr><tr><td>Silicone spacers (round, 24.2 mm outer diameter, 14.3 mm inner diameter, 1.8 mm depth)</td><td></td><td></td><td>laboratory-made, cut from silicone sheet</td></tr><tr><td>Soy PC (95%)</td><td>Avanti Research</td><td>441601</td><td></td></tr><tr><td>Specord 50 PLUS spectrophotometer</td><td>Analytik Jena</td><td>823-0200P-3</td><td></td></tr><tr><td>Superdex 200 10/300 GL column (column L &amp;times; I.D. 30 cm &amp;times; 10 mm)</td><td>Cytiva</td><td>28-9909-44</td><td></td></tr><tr><td>Test tube shaker (2800 rpm)</td><td>IKA Lab Dancer</td><td>3365000</td><td></td></tr><tr><td>Thermomixer comfort 5355</td><td>Eppendorf</td><td>5355000.011</td><td></td></tr><tr><td>Xiameter OFX-5329 (PDMS-g-PEO; MW 3000)</td><td>Dow Corning</td><td>000000000004109892</td><td></td></tr><tr><td>Zetasizer Nano ZS&amp;nbsp;</td><td>Malvern Panalytical</td><td>ZEN3600</td><td></td></tr></tbody></table>

preparation of membrane protein&#8211;functionalized polymer and polymer/lipid hybrid large and giant unilamellar vesicles

Preparation of Membrane Protein&#8211;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&#8211;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&#8211;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&#8211;35 &#181;m 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 &#177; 3.7 a.u. for polymer GUVs and 26.2 &#177; 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&#8217;s mechanical softness and lipid-like bilayer thickness (~5.3 nm) support efficient protein insertion and preservation of functionality.

The E. coli cytochrome bo3 oxidase was expressed from plasmid pETcyo in E. coli strain C43 (DE3) &#916;cyoABCDE and purified as described previously29. Purified bo3 oxidase was either used directly for reconstitution and activity measurements, or labeled with fluorescent dyes, such as ATTO 643 (this study) or other ATTO dyes1,15. For highly hydrophilic dyes like ATTO 643, a 30 cm co...

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&#8211;electroformation. The resulting functional polymer-based compartments are suitable for advanced biochemical and biophysical studies.

Vesicles formed from amphiphilic copolymers, alone or blended with phospholipids, offer superior mechanical and chemical stability compared to ...

Our research focuses on building artificial cells from the bottom up, combining natural and synthetic materials to form fully functional biomimetic systems. Our protocol for building protein-functionalized polymer vesicles applies to drug delivery, construction of artificial cells, nanoreactors and biosensors.To begin, use positive-displacement pipettes with solvent-resistant tips or gas-tight glass syringes to transfer the polymer, lipid and fluorescent dye solutions dissolved in a 2-to-1 mixture of chloroform and methanol into a clean glass vial. Mix the solution thoroughly by gentle vortexing while holding the glass vial upright to ensure homogeneity. Evaporate the organic solvents under a gentle stream of nitrogen in the fume hood for approximately one hour, until a uniform thin film forms on the glass vial wall. If polymersomes are used for reconstitution, supplement the rehydration buffer with 0.1% sodium cholate, then rehydrate the dried film with 1 milliliter of rehydration buffer to obtain a final amphiphile concentration of 5mg/ml. Vortex the glass vial vigorously until the film is completely detached and suspended, forming multilamellar vesicles. Subject the hybrid multi

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Learn to prepare membrane protein-functionalized polymer and hybrid vesicles using detergent-mediated reconstitution. This protocol enables stable LUV and GUV formation for drug delivery, biosensing, and artificial cell applications.

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

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