APL acknowledges support from the National Science Foundation (EF1935265), the National Institutes of Health (R01-EB030031 and R21-AR080363), and the Army Research Office (80523-BB)

The reagents and equipment utilized for this study are provided in the Table of Materials.
1. Bulk CFE reactions in the presence of small unilamellar vesicles (SUVs)
<ol>
	<li>SUV preparation 
	NOTE: This step needs to be performed in a fume hood following the safety instructions for working with chloroform.
	<ol>
		<li>Prepare 5 mM 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) SUVs in a glass vial by transferring 76 &#181;L of 25 mg/mL POPC stock solution dissolved in chloroform.</li>
		<li>While gently rotating the glass vial, blow a gentle stream of argon into the vial to form a film of dried lipids at the bottom. Next, transfer the glass vial to a desiccator with its cap loosely screwed to evaporate excess chloroform.</li>
		<li>Keep the glass vial in the desiccator for 1 h. Then, add 0.5 mL ultra-pure deionized water to dissolve the lipid film and vortex for approximately 2 min.</li>
		<li>Set up a mini-extrusion apparatus by soaking two filter supports in deionized water and placing them on each of the internal membrane supports. Then, soak a 100 nm polycarbonate filter and place it in between the two internal membrane supports held together by the extruder outer casing and the retainer nut. Place this setup in the extruder stand.</li>
		<li>Flush two 1 mL gas-tight syringes 3 times with ultra-pure deionized water.</li>
		<li>Load the sample of lipid-water mix into one of the 1 mL gas-tight syringes and place it in one end of the mini extruder using the swing arm clips to hold the syringe in place. Insert the second syringe into the other end of the mini-extruder and make sure it is fully depressed. 
		NOTE: When loading the syringe with the lipid-water mix, ensure there is no air in the syringe before passing it through the mini-extruder.</li>
		<li>Gently pass the lipid-water mix from the original syringe to the empty syringe through the mini-extruder apparatus. Repeat this step 11 times to form SUVs. Transfer the SUVs to a 1.5 mL microcentrifuge tube. 
		NOTE: The SUV solution can be stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>CFE reaction assembly
	<ol>
		<li>Assemble the CFE reaction by following the cell-free expression protocol provided by the manufacturer with slight modifications detailed in the following.</li>
		<li>Mix 10 &#181;L of Solution 1 (containing amino acids, NTPs, tRNAs and substrates for enzymes, and necessary buffer), 1 &#181;L of Solution 2 (proteins in 20% glycerol), 2 &#181;L of Solution 3 (ribosome (20 &#181;M)), appropriate amount of the DNA encoding for soluble sfGFP-sfCherry(1-10)27&#160;(referred to as soluble sfGFP hereafter), WT-GluR0-sfGFP, or PRSP-GluR0-sfGFP (10-60 ng/1000 base pairs), 1 &#181;L murine RNAse inhibitor, and 4 &#181;L of 5 mM SUV solution for membrane proteins or 4 &#181;L water for soluble proteins.</li>
		<li>Bring the reaction&#39;s final volume to 20 &#181;L by adding ultra-pure deionized water. 
		NOTE: When assembling the CFE system, all components must be kept on ice. All materials are temperature-sensitive and can degrade if they reach room temperature.</li>
	</ol>
	</li>
	<li>CFE reaction incubation and monitoring
	<ol>
		<li>Transfer the CFE solution to a 96-well conical V-bottom plate. To prevent evaporation during the course of the reaction, cover the plate using a sealing film.</li>
		<li>Incubate the plate at 37 &#176;C in a plate reader for 4-5 h while monitoring the CFE reaction by measuring the GFP signal with a gain of 100 at 488 nm/528 nm excitation/emission wavelengths every 2 min.</li>
	</ol>
	</li>
</ol>
2. CFE reactions encapsulated in GUVs
<ol>
	<li>Preparation of GUV outer buffer solution
	<ol>
		<li>In a 1.5 mL microcentrifuge tube, mix 1.5 &#181;L of 1 M spermidine, 37.5 &#181;L of 100 mM ATP, 25 &#181;L of 100 mM GTP, 12.5 &#181;L of 100 mM CTP, 12.5 &#181;L of 100 mM UTP, 25 &#181;L of 1 M creatine phosphate, 18 &#181;L of 1 M magnesium acetate, 93.33 &#181;L of 3 M potassium glutamate, 50 &#181;L of 1 M HEPES KOH (pH 7.4), 1.15 &#181;L of 332 mM folinic acid, 100 &#181;L of 2 M glucose, and 50 &#181;L from stock solution of 6 mM mixture of each of the 20 amino acids (prepared by following the protocol described by Sun et al.28).</li>
		<li>Bring the solution&#39;s final volume to 1 mL by adding ultra-purified deionized water. 
		NOTE: All components must be kept on ice. Add the amino acid mixture at the end to avoid amino acid depletion28. The solution can be aliquoted in 330 &#181;L aliquots and stored at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Preparation of the lipid-in-oil mixture 
	NOTE: this step needs to be performed in a fume hood following the safety instructions for working with chloroform.
	<ol>
		<li>In a fume hood, mix 17.3 &#181;L of 25 mg/mL POPC stock solution and 1.08 &#181;L of 50 mg/mL poly(butadiene)-b-poly(ethylene oxide) (PEO-b-PBD) copolymer in a 15 mL glass vial. 
		NOTE: The final lipid-in-oil solution contains 0.5 mM lipid with 95% and 5% POPC and PEO-b-PBD, respectively. PEO-b-PBD was used to enhance membrane stability during protein expression but was kept at a low molar ratio to reduce the copolymer&#39;s tendency to aggregate into micelles separate from lipid molecules29,30.</li>
		<li>Carefully blow a gentle stream of argon gas into the glass vial while rotating the vial to evaporate the chloroform.</li>
		<li>Pipette 1.2 mL of light mineral oil into the 15 mL glass vial containing the dried lipids.</li>
		<li>Mix the lipids and oil by vortexing at maximum speed for 10-20 s. The dissolved lipid-copolymer mix will look cloudy.</li>
		<li>To ensure that all possible lipid aggregates in the oil are fully dissolved and dispersed throughout the oil, place the glass vial in an oven at around 50 &#176;C for 20 min before vortexing for an additional 10-20 s at maximum speed.</li>
	</ol>
	</li>
	<li>CFE reaction assembly and encapsulation (Figure 1 summarizes the following steps).
	<ol>
		<li>Prepare 300 &#181;L of the GUV outer solution in a 1.5 mL microcentrifuge tube by mixing 270 &#181;L of CFE outer buffer solution prepared in step 2.1, 15 &#181;L of 5 M NaCl, and 15 &#181;L of 4.5 M KCl. 
		NOTE: At this step, add 0.45 &#181;L of 1 M 1,4-dithiothreitol (DTT) to the GUV outer solution. The purpose of adding NaCl and KCl is to adjust the outer solution&#39;s osmolality to match it with the inner CFE solution. The exact volume of NaCl and KCl depends on the desired osmolality adjustment. One can add either NaCl or KCl or both to adjust the osmolality.</li>
		<li>Gently pipette 300 &#181;L of lipid-in-oil mixture on top of the GUV outer solution. 
		NOTE: When adding the lipid-oil mix, it is important that the oil does not mix with the aqueous outer solution. After the addition, there should be a visible interface between the lipid-in-oil mixture and the GUV outer solution.</li>
		<li>Incubate the oil-water interface at room temperature for 2 h to allow the lipid monolayer to form and stabilize at the interface.</li>
		<li>Meanwhile, follow section 1.2.1 to assemble a CFE reaction containing the plasmid DNA encoding the membrane protein variants or soluble GFP. Replace the 4 &#181;L of 5 mM SUV solution or water with 4 &#181;L of 1 M sucrose. This reaction will be the GUV inner solution. 
		NOTE: An osmometer was used to measure the osmolality of the inner and outer solutions. The osmolality of the outer solution was then adjusted accordingly by the addition of NaCl or water. The osmolality of the CFE reaction is typically around 1600 mOsm/Kg. Adding sucrose to the reaction increases the inner solution density, thus allowing the vesicles to travel across the oil-water interface during the centrifugation step. An alternative to sucrose is the Opti-Prep density gradient solution.</li>
		<li>Add 600 &#181;L of lipid-oil mixture to the microcentrifuge tube containing the CFE reaction and pipette up and down vigorously for ~1 min to emulsify the reaction in lipid-in-oil solution and form the lipid monolayer around synthetic cells. 
		NOTE: The final solution should not have any bubbles and should look opaque.</li>
		<li>Gently pipette the inner solution emulsion on top of the oil layer in the 1.5 mL microcentrifuge tube where the oil-water interface was set up. 
		NOTE: Be careful not to disturb or destabilize the interface.</li>
		<li>Centrifuge for 10 min at 2,000 x g at 4 &#176;C. 
		NOTE: The centrifugation speed was optimized for this protocol. Adir et al.31 reported a different centrifugation speed.</li>
		<li>Once the centrifugation is over, carefully remove the excess oil and outer solution from the microcentrifuge tube using a pipettor. Remove the outer solution until the remaining volume is around 100 &#181;L. 
		NOTE: A pellet of GUVs is usually visible at the bottom of the microcentrifuge tube. However, a lack of a visible pellet does not necessarily mean no GUV yield. Instead of using a pipettor, the excess oil on top of the outer solution can be removed by aspiration. It is critical to ensure the lipid-in-oil solution is completely removed. Oil contamination in GUV solution can cause low-quality images.</li>
		<li>Resuspend the GUV pellet in the remaining 100 &#181;L solution by gently pipetting up and down. Next, transfer the GUV solution to a clean 96 well clear flat bottom plate to incubate.</li>
	</ol>
	</li>
</ol>
3. Encapsulated CFE reaction incubation and imaging
<ol>
	<li>Cover the plate using a sealing film to prevent evaporation. Incubate the plate at 37 &#176;C for 5-6 h. One can use a plate reader and follow step 1.3.2 to prepare the plate reader for the incubation step.</li>
	<li>Once the incubation is over, place the 96 well plate on the imaging stage of an inverted microscope equipped with an EMCCD camera (or a sCMOS camera), DAQ-MX controlled laser (or an integrated laser combiner system), and a CSU-X1 spinning disk confocal (or a laser scanning confocal). Focus on any ROI containing GUVs and capture images at an excitation wavelength of 488 nm using a Plan-Apochromat 60 x/1.4 NA objective.</li>
	<li>Save images of GUVs in .tiff format.</li>
	<li>Open the images in an image processing software (e.g., ImageJ or Fiji). Open the Brightness/Contrast setting panel. Adjust the brightness and contrast to appropriate settings that make fluorescent proteins visible.</li>
	<li>If the goal is to compare the signal intensity of different expressed proteins, first stack individual images of GUVs containing different proteins using Images to Stack panel located under Image &#62; Stacks submenu. Then, adjust the brightness and contrast of all images using the Brightness/Contrast panel.</li>
</ol>

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

<ol>
	<li>Liu, A. P., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+under+construction:+In+vitro+reconstitution+of+cellular+function.">Biology under construction: In vitro reconstitution of cellular function.</a> Nat Rev Mol Cell Biol. 10 (9), 644-650 (2009).</li><li>Lin, A. J., Sihorwala, A. Z., Belardi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+tissue-scale+properties+with+synthetic+cells:+Forging+one+from+many.">Engineering tissue-scale properties with synthetic cells: Forging one from many.</a> ACS Synth Biol. 12 (7), 1889-1907 (2023).</li><li>Powers, J., Jang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+biomimetic+functions+of+synthetic+cells+through+compartmentalized+cell-free+protein+synthesis.">Advancing biomimetic functions of synthetic cells through compartmentalized cell-free protein synthesis.</a> Biomacromolecules. 24 (12), 5539-5550 (2023).</li><li>Jiang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+cells:+Past,+present+and+future.">Artificial cells: Past, present and future.</a> ACS Nano. 16 (10), 15705-15733 (2022).</li><li>Groaz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+spatiotemporal+organization+and+dynamics+in+synthetic+cells.">Engineering spatiotemporal organization and dynamics in synthetic cells.</a> WIREs Nanomed Nanobiotech. 13 (3), 1685 (2021).</li><li>Sharma, B., Moghimianavval, H., Hwang, S. W., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+cell+as+a+platform+for+understanding+membrane-membrane+interactions.">Synthetic cell as a platform for understanding membrane-membrane interactions.</a> Membranes. 11 (12), 912 (2021).</li><li>Bashirzadeh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+crosslinker+competition+and+sorting+drive+emergent+GUV+size-dependent+actin+network+architecture.">Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture.</a> Commun Biol. 4 (1), 1-11 (2021).</li><li>Bashirzadeh, Y., Moghimianavval, H., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+actomyosin+patterns+drive+cell-like+membrane+shape+changes.">Encapsulated actomyosin patterns drive cell-like membrane shape changes.</a> iScience. 25 (5), 104236 (2021).</li><li>Berhanu, S., Ueda, T., Kuruma, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+photosynthetic+cell+producing+energy+for+protein+synthesis.">Artificial photosynthetic cell producing energy for protein synthesis.</a> Nat Commun. 10 (1), 1325 (2019).</li><li>Ji, Y., Chakraborty, T., Wegner, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-regulated+and+bidirectional+communication+in+synthetic+cell+communities.">Self-regulated and bidirectional communication in synthetic cell communities.</a> ACS Nano. 17 (10), 8992-9002 (2023).</li><li>Moghimianavval, H., Loi, K. J., Hwang, S. W., Bashirzadeh, Y., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-based+juxtacrine+signaling+between+synthetic+cells.">Light-based juxtacrine signaling between synthetic cells.</a> bioRxiv. , (2024).</li><li>Boyd, M. A., Thavarajah, W., Lucks, J. B., Kamat, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+and+tunable+performance+of+a+cell-free+biosensor+encapsulated+in+lipid+vesicles.">Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles.</a> Science Advances. 9 (1), 6605 (2023).</li><li>Schneider, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Protein+expression+in+cell-free+systems.">Membrane Protein expression in cell-free systems.</a> Methods Mol Biol. 601, 165-186 (2010).</li><li>Noireaux, V., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+age+of+cell-free+biology.">The new age of cell-free biology.</a> Annu Rev Biomed Eng. 22 (1), 51-77 (2020).</li><li>Majumder, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-sized+mechanosensitive+and+biosensing+compartment+programmed+with+DNA.">Cell-sized mechanosensitive and biosensing compartment programmed with DNA.</a> Chem. Commun. 53 (53), 7349-7352 (2017).</li><li>Poddar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+stretching+activates+calcium+permeability+of+a+putative+channel+Pkd2+during+fission+yeast+cytokinesis.">Membrane stretching activates calcium permeability of a putative channel Pkd2 during fission yeast cytokinesis.</a> MBoC. 33 (14), (2022).</li><li>Sachse, R., Dondapati, S. K., Fenz, S. F., Schmidt, T., Kubick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+synthesis+in+cell-free+systems:+From+bio-mimetic+systems+to+bio-membranes.">Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes.</a> FEBS Letters. 588 (17), 2774-2781 (2014).</li><li>Dondapati, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+membrane+proteins+derived+from+eukaryotic+cell-free+systems.">Functional reconstitution of membrane proteins derived from eukaryotic cell-free systems.</a> Front Pharmacol. 10, 917 (2019).</li><li>Majumder, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+and+reconstitution+using+mammalian+cell-free+lysates+enables+the+systematic+study+of+the+regulation+of+LINC+complex+assembly.">In vitro synthesis and reconstitution using mammalian cell-free lysates enables the systematic study of the regulation of LINC complex assembly.</a> Biochemistry. 61 (14), 1495-1507 (2022).</li><li>Niwa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+study+of+liposome-assisted+synthesis+of+membrane+proteins+using+a+reconstituted+cell-free+translation+system.">Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system.</a> Sci Rep. 5 (1), 18025 (2015).</li><li>Moghimianavval, H., Hsu, Y. Y., Groaz, A., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+platforms+of+mammalian+cell-free+expressed+membrane+proteinsmembrane+proteins.">In vitro reconstitution platforms of mammalian cell-free expressed membrane proteinsmembrane proteins.</a> Methods Mol Biol. 2433, 105-120 (2022).</li><li>Eaglesfield, R., Madsen, M. A., Sanyal, S., Reboud, J., Amtmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+recruitment+of+ribosomes+in+protocells+recreates+a+translocon-independent+mechanism+of+proteorhodopsin+biogenesis.">Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis.</a> iScience. 24 (5), 102429 (2021).</li><li>Steink&#252;her, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cell-free+expression+of+membrane+proteins+by+tuning+ribosome+cotranslational+membrane+association+and+nascent+chain+aggregation.">Improving cell-free expression of membrane proteins by tuning ribosome cotranslational membrane association and nascent chain aggregation.</a> bioRxiv. , (2023).</li><li>Chen, G. Q., Cui, C., Mayer, M. L., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+a+potassium-selective+prokaryotic+glutamate+receptor.">Functional characterization of a potassium-selective prokaryotic glutamate receptor.</a> Nature. 402 (6763), 817-821 (1999).</li><li>Pautot, S., Frisken, B. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+unilamellar+vesicles+using+an+inverted+emulsion.">Production of unilamellar vesicles using an inverted emulsion.</a> Langmuir. 19 (7), 2870-2879 (2003).</li><li>Hsu, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-triggered+DNA-mediated+membrane+fusion+in+synthetic+cells.">Calcium-triggered DNA-mediated membrane fusion in synthetic cells.</a> Chemical Communications. 59 (57), 8806-8809 (2023).</li><li>Moghimianavval, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+functional+membrane-membrane+interfaces+by+interspy.">Engineering functional membrane-membrane interfaces by interspy.</a> Small. 19 (13), 2202104 (2023).</li><li>Sun, Z. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> J Vis Exp. (79), e50762 (2013).</li><li>Jacobs, M. L., Boyd, M. A., Kamat, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diblock+copolymers+enhance+folding+of+a+mechanosensitive+membrane+protein+during+cell-free+expression.">Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression.</a> PNAS. 116 (10), 4031-4036 (2019).</li><li>Kostarelos, K., Tadros, T. F., Luckham, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+conjugation+of+(tri-)+block+copolymers+to+liposomes+toward+the+construction+of+sterically+stabilized+vesicle+systems.">Physical conjugation of (tri-) block copolymers to liposomes toward the construction of sterically stabilized vesicle systems.</a> Langmuir. 15 (2), 369-376 (1999).</li><li>Adir, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+protein+producing+synthetic+cells+using+cell+free+bacterial+extracts,+liposomes+and+emulsion+transfer.">Preparing protein producing synthetic cells using cell free bacterial extracts, liposomes and emulsion transfer.</a> J Vis Exp. (158), e60829 (2020).</li><li>van de Cauter, L., van Buren, L., Koenderink, G. H., Ganzinger, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+giant+unilamellar+vesicle+production+for+artificial+cells+-+current+challenges+and+future+directions.">Exploring giant unilamellar vesicle production for artificial cells - current challenges and future directions.</a> Small Methods. 7 (12), 2300416 (2023).</li><li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins,+lipids+and+detergents:+Not+just+a+soap+opera.">Membrane proteins, lipids and detergents: Not just a soap opera.</a> Biochim Biophys Acta Biomembr. 1666 (1), 105-117 (2004).</li><li>Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent-free+systems+for+structural+studies+of+membrane+proteins.">Detergent-free systems for structural studies of membrane proteins.</a> Biochem Soc Trans. 49 (3), 1361-1374 (2021).</li><li>Bashirzadeh, Y., Wubshet, N., Litschel, T., Schwille, P., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+encapsulation+of+reconstituted+cytoskeleton+inside+giant+unilamellar+vesicles.">Rapid encapsulation of reconstituted cytoskeleton inside giant unilamellar vesicles.</a> J Vis Exp. (177), e63332 (2021).</li><li>Hwang, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vesicles+enable+mechano-responsive+hydrogel+degradation.">Hybrid vesicles enable mechano-responsive hydrogel degradation.</a> Angew Chemie Int Ed. 62 (41), e202308509 (2023).</li><li>Rideau, E., Dimova, R., Schwille, P., Wurm, F. R., Landfester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+polymersomes:+a+comparative+review+towards+cell+mimicking.">Liposomes and polymersomes: a comparative review towards cell mimicking.</a> Chem Soc Rev. 47 (23), 8572-8610 (2018).</li><li>Tsumoto, K., Hayashi, Y., Tabata, J., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reverse-phase+method+revisited:+Rapid+high-yield+preparation+of+giant+unilamellar+vesicles+(GUVs)+using+emulsification+followed+by+centrifugation.">A reverse-phase method revisited: Rapid high-yield preparation of giant unilamellar vesicles (GUVs) using emulsification followed by centrifugation.</a> Colloids Surf A: Physicochem Eng Asp. 546, 74-82 (2018).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> J Am Chem Soc. 137 (33), 10767-10776 (2015).</li><li>Bailoni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+out-of-equilibrium+metabolism+for+synthetic+cells:+A+membrane+perspective.">Minimal out-of-equilibrium metabolism for synthetic cells: A membrane perspective.</a> ACS Synth Biol. 12 (4), 922-946 (2023).</li></ol>

The authors declare no conflicts of interest.

Virtually any cellular process that depends on the transfer of molecules or information across the cell membrane, like cell signaling or cell excitation, requires membrane proteins. Thus, the reconstitution of membrane proteins has become the main bottleneck in realizing various synthetic cell designs for different applications. Traditional detergent-mediated reconstitution of membrane proteins in biological membranes requires GUV generation methods such as gentle swelling or electroformation. Swelling approaches usually...

Bottom-up synthetic biology has gained increasing interest over the past decade as an emerging field with numerous potential applications in bioengineering, drug delivery, and regenerative medicine1,2. The development of synthetic cells as a cornerstone of bottom-up synthetic biology, in particular, has attracted a wide range of scientific communities due to the promising applications of synthetic cells as well as their cell-like physical and biochemical properties that facilitate in vitro biophysical studies3,4,5,6. Synthetic cells are often engineered in cell-sized giant unilamellar vesicles (GUVs) in which different biological processes are recreated. Reconstitution of cell cytoskeleton7,8, light-dependent energy regeneration9, cellular communication10,11, and biosensing12 are examples of efforts made to reconstruct cell-like behaviors in synthetic cells.
While some cellular processes rely on soluble proteins, many characteristics of natural cells, such as sensing and communication, often utilize membrane proteins, including ion channels, receptors, and transporters. A major challenge in synthetic cell development is the reconstitution of membrane proteins. Although traditional methods of membrane protein reconstitution in lipid bilayers rely on detergent-mediated purification, such methods are laborious, ineffective for proteins that are toxic to the expression host, or are often not suited for membrane protein reconstitution in GUVs13.
An alternative method for protein expression is cell-free expression (CFE) systems. CFE systems have been a powerful tool in synthetic biology that allows in vitro expression of various proteins using either cell lysate or purified transcription-translation machinery14. CFE systems can also be encapsulated in GUVs, thus allowing compartmentalized protein synthesis reactions that can be programmed for various applications, such as the creation of light-harvesting synthetic cells9 or mechanosensitive biosensors15,16. Analogous to recombinant protein expression methods, membrane protein expression is challenging in CFE systems17. Aggregation, misfolding, and lack of post-translational modification in CFE systems are major bottlenecks that hinder successful membrane protein synthesis using CFE systems. The difficulty of bottom-up membrane protein reconstitution using CFE systems is due in part to the absence of a complex membrane protein biogenesis pathway that relies on signal peptides, signal recognition particles, translocons, and chaperoning molecules. However, recently, multiple studies have suggested that the presence of membranous structures such as microsomes or liposomes during translation promotes successful membrane protein expression18,19,20,21. Additionally, Eaglesfield et al. and Steink&#252;her et al. have found that the inclusion of specific hydrophobic domains known as signal peptides in the N-terminus of the membrane protein can significantly improve its expression22,23. Altogether, these studies suggest that the challenge of membrane protein reconstitution in synthetic cells can be overcome if the protein translation occurs in the presence of the GUV membrane and if proper N-terminal signal peptide is utilized.
Here, a protocol for encapsulation of the protein synthesis using recombinant elements (PURE) CFE reactions for membrane protein reconstitution in GUVs is presented. Bacterial glutamate receptor24 (GluR0) is selected as the model membrane protein, and the effect of its N-terminal signal peptide on its membrane reconstitution is studied. The effect of proteorhodopsin signal peptide, which was shown to improve membrane protein reconstitution efficiency by Eaglesfield et al.22, is investigated by constructing a mutated variant of GluR0 denoted as PRSP-GluR0 and its expression and membrane localization with wild-type GluR0 (referred to as WT-GluR0 hereafter) that harbors its native signal peptide is compared. This protocol is based on the inverted emulsion method25 with modifications that make it robust for CFE encapsulation. In the presented method, the CFE reactions are first emulsified using a lipid-in-oil solution that generates micron-sized droplets that contain the CFE system and are stabilized by the lipid monolayer. The emulsion droplets are then layered on top of an oil-water interface that is saturated with another lipid monolayer. The emulsion droplets are then forced to travel across the oil-water interface via centrifugal force. Through this process, the droplets obtain another monolayer, thus generating a bilayer lipid vesicle. The GUVs containing the CFE reaction are then incubated, during which the membrane protein is expressed and incorporated into the GUV membrane. Although this protocol is specified for cell-free expression of GluR0, it can be used for cell-free synthesis of other membrane proteins or different synthetic cell applications such as cytoskeleton reconstitution or membrane fusion studies26.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 nm polycarbonate filter</td>
			<td>STERLITECH</td>
			<td>1270193</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Clear Bottom Plate</td>
			<td>ThermoFisher Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy H1M Hybrid Multi-Mode Reader</td>
			<td>Agilent</td>
			<td>11-120-533</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Millipore Sigma</td>
			<td>10621714001</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1 Confocal Scanner Unit</td>
			<td>Yokogawa</td>
			<td>CSU-X1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium (Optiprep)</td>
			<td>Millipore Sigma</td>
			<td>D1556</td>
			<td>Optional to switch with sucrose in inner solution</td>
		</tr>
		<tr>
			<td>Filter supports</td>
			<td>Avanti</td>
			<td>610014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand microtubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-129&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Millipore Sigma</td>
			<td>F7878</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>158968</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>iXon X3 camera&#160;</td>
			<td>Andor</td>
			<td>DU-897E-CS0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid potassium salt monohydrate</td>
			<td>Millipore Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Millipore Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate tetrahydrate&#160;</td>
			<td>Millipore Sigma</td>
			<td>M5661</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extruder kit (including syringe holder and extruder stand)</td>
			<td>Avanti</td>
			<td>610020</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX81 Inverted Microscope&#160;</td>
			<td>Olympus</td>
			<td>IX21</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus PlanApo N 60x Oil Microscope Objective&#160;</td>
			<td>Olympus</td>
			<td>1-U2B933&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-b-PBD</td>
			<td>Polymer Source</td>
			<td>P41745-BdEO</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-PRSP-GluR0-sfGFP plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-sfGFP-sfCherry(1-10) plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-WT-GluR0-sfGFP plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC lipid in chloroform&#160;</td>
			<td>Avanti</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>PUREfrex 2.0</td>
			<td>Cosmo Bio USA</td>
			<td>GFK-PF201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleotide Solution Set</td>
			<td>New England BioLabs</td>
			<td>N0450</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor, Murine</td>
			<td>New England BioLabs</td>
			<td>M0314S</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS Amino Acid Sampler</td>
			<td>Biotechrabbit</td>
			<td>BR1401801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Millipore Sigma</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>VAPRO Vapor Pressure Osmometer Model 5600</td>
			<td>ELITechGroup</td>
			<td>VAPRO 5600</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of the bacterial glutamate receptor channel by encapsulation of a cell-free expression system

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Our protocol addresses the reconstitution of a membrane protein within the lipid bilayer of a synthetic cell made using the inverted droplet emulsion method. The advantage of using the inverted droplet emulsion method is its ability to generate synthetic cells that provide a stable environment for the cell-free expression of membrane proteins. Our protocol offers a robust method for researchers in the field to synthesize membrane proteins inside synthetic cells using cell-free expression system.My lab is working on reconstituting various ion channels in synthetic cells through the reconstitution of the action potential by a well-defined set of ion channels.

Prior to encapsulation of the CFE reactions, two variants of GluR0-sfGFP harboring native and proteorhodopsin signal peptides (signal peptide sequences are presented in Supplementary&#160;Table 1), and the soluble sfGFP were individually expressed in bulk reactions, and their expression was monitored by detecting the sfGFP signal using a plate reader (Figure 2A). Membrane proteins were expressed in the absence or presence of 100 nm SUVs. Additionally, using ...

Watch this Scientific Journal Video about Membrane Protein Reconstitution in Synthetic Cells at JoVE.com

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

reconstitution-bacterial-glutamate-receptor-channel-encapsulation

Research

JoVE Journal

Bioengineering

787 Views.  University of Michigan, Ann Arbor. Our protocol addresses the reconstitution of a membrane protein within the lipid bilayer of a synthetic cell made using the inverted droplet emulsion method. The advantage of using the inverted droplet emulsion method is its ability to generate synthetic cells that provide a stable environment for the cell-free expression of membrane proteins. Our protocol offers a robust method for researchers in the field to synthesize membrane proteins inside synthetic cells using cell-free expression syst...

Video: Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

Bulk Cell-Free Expression Reactions with Small Unilamellar Vesicles (SUVs) for Synthetic Cell Applications

To begin, blow a gentle stream of Argonne into a glass vial containing POPC lipids while gently rotating the vial. When the lipids have dried as a film at the bottom of the vial, loosely cap it, then transfer it to a desiccater. After one hour add 0.5 milliliters of ultrapure deionized water to dissolve the lipid film.Vortex the solution for two minutes. Next, soak two filter supports and deionized water. Place them on each of the internal membrane supports.Then place a 100 nanometer polycarbonate filter between the two internal membrane supports, held together by the extruder outer casing and retainer nut. Place the setup in the extruder stand to finish setting up the mini extrusion apparatus. Next, load the lipid water mix into a one milliliter gast tight syringe.Use swing arm clips to secure the syringe into one end of the mini extruder. Insert the second syringe into the other end of the mini extruder, making sure that it is fully depressed. Now pass the lipid water mix from the original syringe into the empty one through the apparatus.After repeating the extrusion 11 times, transfer the SUVs to a 1.5 milliliter micro centrifuge tube. To assemble the cell-free expression protocol, pipette solutions one to 3 DNA plasmid encoding for soluble SFGFP or variants of glutamate receptor SFGFP, murine RNAse, and SUV solution into a vial. Add ultrapure deionized water to bring the reaction volume to 20 microliters.Transfer the cell-free expression solution to a 96 well conical V bottom plate. Incubate the plate in a plate reader at 37 degrees Celsius for four to five hours. Set up the plate reader with a gain of 100, excitation of 485 nanometers, emission of 528 nanometers, a runtime of five hours, and an interval measurement of one read for every two minutes.Measure the GFP signal every two minutes to monitor the cell-free reaction in real time. Soluble SFGFP had the highest expression among all three proteins.

Cell-Free Reactions in Giant Unilamellar Vesicles (GUVs) for Synthetic Cell Applications

To begin, prepare the GUV outer buffer solution in a 1.5-milliliter microcentrifuge tube. Mixed together spermidine, ATP, GTP, CTP, UTP, creatine phosphate, magnesium acetate, potassium glutamate, HEPES, potassium hydroxide, folinic acid. glucose, and amino acid stock.Add ultra purified deionized water to bring the final volume of the solution to one milliliter. Next, in a fume hood, add 17.3 microliters of POPC stock solution to a 15-milliliter glass vial. To this, pipette 1.08 microliters of PEO-b-PBD copolymer.Carefully blow a gentle stream of argon gas into the glass vial while rotating the vial to evaporate the chloroform. Then pipette 1.2 milliliters of light mineral oil into the glass vial. Vortex the lipid and oil at maximum speed for 10 to 20 seconds.Place the glass vial in an oven at around 50 degrees Celsius for 20 minutes before vortexing for an additional 10 to 20 seconds at maximum speed. Next, pipette 270 microliters of the GUV outer solution into a 1.5-milliliter microcentrifuge tube. To this, pipette 15 microliters each of five molar sodium chloride and 4.5 molar potassium chloride.Gently pipette 300 microliters of lipid and oil mixture on top of the GUV outer solution. After incubation, assemble a CFE reaction by pipetting Solutions 1 to 3, DNA plasmid encoding for soluble sfGFP or variants of glutamate receptor sfGFP, murine RNAse, and one molar sucrose into a vial. Add ultrapure deionized water to bring the reaction volume to 20 microliters.Add 600 microliters of the lipid-oil mixture to the microcentrifuge tube containing the 20-microliter reaction mixture. Pipette up and down vigorously for one minute to emulsify the reaction. Gently layer the inner emulsion on top of the oil layer in the tube.Centrifuge for 10 minutes at 2000 g at four degrees Celsius. Next, carefully pipette out the excess oil and outer solution from the microcentrifuge tube. Gently mix the GUV pellet in the remaining solution to resuspend it.Transfer the GUV solution to a clean 96-well clear flat bottom plate for incubation. Transfer the sealed plate into a plate reader to incubate at 37 degrees Celsius for five to six hours. After incubation, place the plate on an inverted confocal microscope.Focus on a region of interest containing the GUVs, then capture the images at an excitation wavelength of 488 nanometers. Save the images in the TIFF format. Open the images in an image processing software.Click on the Brightness/Contrast setting panel to open it. Then adjust the brightness and contrast to make the fluorescent proteins visible. The wild-type glutamate receptor cell-free expression reactions encapsulated in GUV demonstrated excellent expression and membrane localization.PRSP-glutamate receptor was prone to aggregation and punctate formation. The soluble sfGFP was expressed in GUVS and stayed in the GUV lumen.