This research was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP20am0101077. This work was also partially supported by JSPS KAKENHI Grant Number 20K05709.

1. Preparation of pEU expression plasmid
NOTE: pEU expression plasmid should include start codon, open reading frame of target membrane protein, and stop codon in the fragment (see Figure 1). Add detection/purification tag sequence(s) at the appropriate position when required. Either restriction enzyme digestion or seamless cloning is applicable for subcloning. Here we describe a protocol using a seamless cloning method.
<ol>
	<li>Prepare insert DNA fragment.
	<ol>
		<li>Amplify the gene of interest by PCR using the cDNA template, primer 1 and primer 2. Primer 1 and primer 2 contain 15 bp overlaps for seamless cloning, respectively (see the Table of Materials). 
		NOTE: Do not include sequences to be processed and removed from mature protein in the cells (e.g., signal sequence). Processing of synthesized protein is not performed in wheat cell-free system. Do not add Kozak sequence. pEU-E01-MCS vector has E01 translation enhancer.</li>
		<li>Add 1/25 volume of DpnI restriction enzyme to the PCR product to remove template plasmid DNA. Incubate for 30 min at 37 &#176;C.</li>
		<li>Use a PCR purification kit to purify the PCR product and adjust concentration at 20&#8211;50 ng/&#181;L.</li>
	</ol>
	</li>
	<li>Linearize pEU-E01-MCS vector.
	<ol>
		<li>Conduct inverse PCR using pEU-E01-MCS, primer 3, and primer 4.</li>
		<li>Add 1/25 volume of DpnI restriction enzyme to the inverse PCR product. Incubate for 30 min at 37 &#176;C.</li>
		<li>Use a PCR purification kit to purify the PCR product as per the manufacturer&#8217;s recommendation. Adjust the concentration at 20&#8211;50 ng/&#181;L.</li>
	</ol>
	</li>
	<li>Mix 2 &#181;L of insert DNA fragment, 2 &#181;L of linearized vector, and 4 &#181;L of 2x seamless cloning enzyme mixture.</li>
	<li>Transform Escherichia coli strain JM109 with the seamless cloning product. Using a spreader, spread the bacterial suspension on a LB-ampicillin agar plate. 
	NOTE: pEU vector has an ampicillin resistance marker.</li>
	<li>Confirm the sequence of the expression plasmid constructed using primer 5 and primer 6 from 5&#8217; and 3&#8217; side of MCS in pEU plasmid, respectively.</li>
	<li>Amplify and purify the expression plasmids.
	<ol>
		<li>Culture the plasmid-transformed E. coli strain JM109 in 150 mL of LB-ampicillin medium at 37 &#730;C and 125 strokes per minute shaking overnight.</li>
		<li>Extract and purify the plasmids using commercially available plasmid prep midi kit. Dissolve plasmids in 500 &#181;L of TE buffer. 
		CAUTION: Do not use a mini prep kit for plasmid extraction. It does not provide sufficient quality and quantity of plasmid.</li>
		<li>Add 500 &#181;L of phenol/chloroform/isoamyl alcohol (25:24:1). Mix vigorously for 5 min, and centrifuge for 5 min at 17,800&#160;x g and room temperature. Transfer the upper plasmid solution to a new tube. 
		CAUTION: Wear disposable gloves to protect the skin from phenol and chloroform. 
		NOTE: In order to remove contamination of RNase from plasmid extraction kit, purify the plasmids using phenol-chloroform purification.</li>
		<li>Add 500 &#181;L of chloroform to remove phenol completely. Mix vigorously for 5 min, and centrifuge for 5 min at 17,800 x g and room temperature. Transfer the upper plasmid solution to a new tube.</li>
		<li>Add 2.5 volume of ethanol and 1/8 volume of 7.5 M ammonium acetate, and store at -30 &#176;C for 1 h.</li>
		<li>Centrifuge at 17,800 x g at 4 &#176;C for 10 min. Wash the pellet with 500 &#181;L of 70% ethanol. Remove the supernatant carefully and leave the pellet for 5 min to dry up.</li>
		<li>Dissolve the expression plasmids in 100 &#181;L of ultrapure water completely. Measure the concentration of plasmids with absorbance at 260 nm. Adjust the concentration to 1 mg/mL.</li>
	</ol>
	</li>
</ol>
2. In vitro transcription
CAUTION: Use DNase and nuclease-free plastic tubes and tips in steps of transcription and translation. Avoid autoclaving plastic wares to prevent contamination.
<ol>
	<li>Harvest ultrapure water in a new plastic tube. 
	CAUTION: Do not use DEPC-treated water because residual DEPC strongly inhibits the reaction. Use freshly purified ultrapure water for transcription and translation.</li>
	<li>Prepare transcription reaction mix by mixing 115.2 &#181;L of ultrapure water, 40 &#181;L of Transcription Buffer LM, 20 &#181;L of NTP mix, 2.4 &#181;L of 80 U/&#181;L RNase inhibitor, 2.4 &#181;L of 80 U/&#181;L SP6 polymerase, and 20 &#181;L of 1 mg/mL pEU expression plasmids. Mix the reagents gently by inverting. Perform a quick spin.</li>
	<li>Incubate the transcription reaction at 37 &#176;C for 6 h.</li>
	<li>Mix the reaction gently by inverting and quickly spin down. Use it immediately for translation, otherwise freeze and store at -80 &#176;C.</li>
	<li>Confirm the transcription product by electrophoresis.
	<ol>
		<li>Mix 100 mL of 1x TAE buffer and 1 g of agarose. Heat the suspension in a microwave oven to prepare 1% agarose TAE gel.</li>
		<li>Take 1 &#181;L of transcription reaction and mix with 3 &#181;L of water and 4 &#181;L of 2x loading dye. 
		NOTE: Denaturing of RNA is not required.</li>
		<li>Load 4 &#181;L of the mixture and 2 &#181;L of DNA ladder marker to the agarose TAE gel.</li>
		<li>Electrophorese at 100 V for 20 min.</li>
		<li>Stain the gel in ethidium bromide for 30 min. Check the ladder band pattern of mRNA using UV transilluminator and gel imager. 
		NOTE: When a smeared band of less than 500 bp is observed, mRNA degradation is suspected.</li>
	</ol>
	</li>
</ol>
3. Preparation of materials for translation
<ol>
	<li>Prepare the translation buffer.
	<ol>
		<li>Mix 27 mL of freshly prepared ultrapure water and 0.75 mL of each 40x stock solution for S1, S2, S3, and S4, respectively in a 50 mL tube. 
		NOTE: Modulate the amounts of materials according to the final required amount of 1x translation buffer. 
		CAUTION: Do not store or refreeze the excess 1x translation buffer after use.</li>
	</ol>
	</li>
	<li>Prepare creatine kinase stock solution. Dissolve lyophilized creatine kinase in ultrapure water to a final concentration of 20 mg/mL. Dispense the solution in small amounts (10 to 50 &#181;L each) in 0.2 mL 8-strip PCR tubes. Freeze the tubes in liquid nitrogen, and store at -80 &#176;C. 
	CAUTION: Do not re-freeze creatine kinase solution after thawing.</li>
	<li>Wash dialysis cups (0.1 mL size) to remove glycerol from the dialysis membrane. 
	NOTE: There are several different-sized dialysis cups. Small-sized cups (0.1 mL) are used for small-scale test (section 5.4), and large-sized cups (2 mL) for large-scale production (section 5.5), respectively. The washing step of dialysis membrane of large-size cups is avoidable.
	<ol>
		<li>Put 1 mL of ultrapure water into a new 1.5 mL tube. Insert small-sized dialysis cup (0.1 mL) to the tube. Add 0.5 mL of ultrapure water into the cup.</li>
		<li>Incubate for more than 30 min at room temperature.</li>
	</ol>
	</li>
</ol>
4. Preparation of liposomes
NOTE: Here we describe two protocols for preparation of liposomes. One uses ready-to-use lyophilized liposomes (section 4.1), while the other produces liposomes by hydrating a thin lipid film (section 4.2).
<ol>
	<li>Prepare liposomes using lyophilized liposomes. 
	NOTE: An easier way to produce proteoliposome is to use commercially available Asolectin liposome. Asolectin is a kind of natural lipid extracted from soybeans.
	<ol>
		<li>Open the vial containing 10 mg of lyophilized Asolectin liposomes (see the Table of Materials) and add 200 &#181;L of translation buffer (section 3.1) to the bottom of the vial. Seal the vial and incubate for 10 min.</li>
		<li>Mix vigorously by putting the vial on the vortex-mixer for 1 min.</li>
		<li>Insert the vial into a 50 mL tube. Spin down the tube by centrifuging at 500 x g for 1 min.</li>
		<li>Using a pipette, transfer the Asolectin liposome suspension (50 mg lipid/mL) to a new 1.5 mL tube. Use liposome immediately for translation, otherwise freeze in liquid nitrogen and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare liposomes by hydrating a thin lipid film.
	<ol>
		<li>If a lipid is sold in powder form, dissolve in chloroform or appropriate organic solvent to 10-100 mg/mL concentration. 
		NOTE: A thin lipid film can be prepared using purified and/or synthesized amphiphilic lipids. The purification method of asolectin is previously described38. Functionally modified lipids, such as biotinylated lipids, fluorescent lipids, and adjuvant lipids, can be added to the basal lipids to produce functional liposomes.</li>
		<li>Transfer the lipid solution containing 50 mg of lipid(s) to an evaporation flask.</li>
		<li>Using a rotary evaporator, evaporate solvent and evenly spread the lipid on the wall of the flask bottom to form a thin film of lipid.</li>
		<li>Put the flask in a vacuum desiccator and leave under negative pressure overnight to remove the solvent completely.</li>
		<li>Add 1 mL of translation buffer to the evaporation flask. Rotate the flask to spread the buffer over the thin lipid film. Incubate for 5 min to hydrate the film.</li>
		<li>Sonicate the flask with an ultrasonic homogenizer or ultrasonic cleaner. Change the angle of the flask occasionally to allow the solution to touch the film thoroughly. Ensure that the thin lipid film is peeled from the bottom and emulsified completely and homogenously. 
		NOTE: Electron micrograph of biotinylated lipids containing liposomes is shown in Figure 1.</li>
		<li>Transfer the liposome suspension (50 mg lipids/mL) to a new 1.5 mL tube. If it is not to be used immediately, freeze the liposomes in liquid nitrogen, and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
5. In vitro translation
<ol>
	<li>Thaw the wheat germ extract quickly by floating the tubes on water at room temperature for a few minutes. After thawing, immediately mix gently by inverting the tubes, spin down, and chill on ice until use. 
	NOTE: Freeze wheat germ extract in liquid nitrogen after use, and store at -80 &#176;C. It withstands several freeze/thaw cycles.</li>
	<li>Thaw 20 mg/mL creatine kinase stock solution. Mix 5 &#181;L of stock solution and 45 &#181;L of translation buffer to prepare 2 mg/mL creatine kinase working solution. 
	CAUTION: Refreezing creatine kinase is not recommended.</li>
	<li>Thaw liposomes or mRNAs when required.</li>
	<li>Conduct a small-scale protein translation.
	<ol>
		<li>Remove water from both the tube and the dialysis cup (0.1 mL), as prepared in step 3.3.2.</li>
		<li>Inject 1 mL and 300 &#181;L of translation buffer in the tube and the dialysis cup, respectively. 
		CAUTION: In case the bottom of the dialysis cup does not reach the surface of translation buffer in the 1.5 mL tube, inject an additional 50&#8211;100 &#181;L of buffer to the tube.</li>
		<li>Prepare translation reaction mixture by mixing 15.6 &#181;L of translation buffer, 2.4 &#181;L of 2 mg/mL creatine kinase, 12 &#181;L of 50 mg/mL liposomes, 15 &#181;L of wheat germ extract, and 15 &#181;L of mRNA. Mix gently by inverting the tubes, and spin down.</li>
		<li>Aspirate 60 &#181;L of the translation reaction mixture using a 200 &#181;L pipette.</li>
		<li>Insert the pipette tip into the undersurface of translation buffer in the dialysis cup. Pipette out the reaction mixture slowly and gently. Cover the dialysis cup with a lid to prevent evaporation. 
		NOTE: The reaction mixture sinks naturally to the bottom of the cup and forms a bilayer. Do not disturb the bilayer by mixing or shaking the cup.</li>
	</ol>
	</li>
	<li>Conduct a large-scale translation (Figure 1).
	<ol>
		<li>Pour 22 mL of translation buffer into a 25 mL tube. Insert a large-sized dialysis cup (2 mL) into the tube and add 2 mL of translation buffer in the cup.</li>
		<li>Prepare a translation reaction mixture by mixing 130 &#181;L of translation buffer, 20 &#181;L of 2 mg/mL creatine kinase, 100 &#181;L of 50 mg/mL liposome, 125 &#181;L of wheat germ extract, and 125 &#181;L of mRNA. Mix gently by pipetting.</li>
		<li>Aspirate all the translation reaction mixture (500 &#181;L) using a 1,000 &#181;L pipette. Inject translation reaction mixture into the dialysis cup in the same way as described in step 5.4.5. Cover dialysis cup with a lid to prevent evaporation.</li>
	</ol>
	</li>
	<li>Incubate the reactions at 15 &#176;C for 24 h.</li>
	<li>Mix the reaction well in the dialysis cup by pipetting. Transfer the crude proteoliposome suspension to a new tube. 
	NOTE: A flat-bottomed 1.5 mL tube is recommended to collect the proteoliposome from the small-scale translation. After centrifugation, liposomes form compact and easily visible pellet on the bottom corner of the tube.</li>
</ol>
6. Purification of proteoliposomes
<ol>
	<li>Centrifuge the tube containing crude proteoliposome suspension at 17,800 x g at 4 &#176;C for 10 min.</li>
	<li>Remove the supernatant. Suspend the proteoliposome pellet in PBS (small scale: 1 mL, large scale: 10 mL) by pipetting.</li>
	<li>Repeat the centrifugation and washing of proteoliposomes for another two circles.</li>
	<li>After washing, add a small amount of PBS and re-suspend proteoliposome pellet well by pipetting. Measure the volume of suspension using a micro pipette. Add PBS to adjust the volume to 60 &#181;L (small scale) or 500 &#181;L (large scale). Transfer the suspension to a new 1.5 mL tube.</li>
	<li>Transfer 10 &#181;L of proteoliposome suspension to a new PCR tube for SDS-PAGE. Divide the rest of the samples into smaller portions for use when necessary. Freeze in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
7. SDS-PAGE and CBB staining
<ol>
	<li>Add 70 &#181;L of water and 40 &#181;L of 3x SDS-PAGE sample buffer to 10 &#181;L of proteoliposome suspension. 
	CAUTION: Do not boil the SDS-PAGE sample, as membrane proteins aggregate, and hardly penetrate into acrylamide gel in electrophoresis. Also, add enough reducing agent to SDS-PAGE sample buffer (e.g., 2-mercaptoethanol at 3% final concentration) to prevent oxidation.</li>
	<li>Set a 5%&#8211;20% gradient SDS-PAGE gel in an electrophoresis chamber. Load 3 &#181;L, 6 &#181;L, 12 &#181;L of proteoliposome samples, 2 &#181;L of protein size marker, and BSA standard series as well.</li>
	<li>Electrophorese at 52 mA, 400 V for 30 min.</li>
	<li>Stain the gel with CBB dye for 1 h. Decolorize in hot water and scan the gel image.</li>
	<li>Using NIH Image J software (https://imagej.nih.gov/ij/), quantify the band intensity of membrane protein in each lane. Estimate the amount of synthesized membrane proteins with BSA standard series.</li>
</ol>

The authors have nothing to disclose.

The presented protocol provides a method of producing membrane proteins at a high success rate. This protocol is simple, highly reproducible, and easy to scale up. It also has the potential to reduce the time and cost of experiments that consume a large amount of membrane proteins. The bilayer-dialysis method improves the productivity by 4&#8211;10 times compared with bilayer method or dialysis method (Figure 2B)45. In an extreme case, the yield of an ion channel and a transporter increased 30 and 20 times, respectively, with bilayer-dialysis method than that with bilayer method (data not shown). High productivity of this protocol is an advantage in antigen production for immunization. Proteoliposomes are often used as immunizing antigens for development of anti-membrane protein antibodies. Highly concentrated and purified membrane proteins imbedded in proteoliposome effectively stimulate immune response and induce antibodies41,47. Using this bilayer-dialysis method, proteoliposomes carrying several milligrams of membrane proteins for immunization purpose can be easily prepared in a few days. Indeed, our group has synthesized GPCRs, ion channels, and claudins using this protocol and immunized mice with the products to obtain monoclonal antibodies against them31,41,45. Some of the monoclonal antibodies obtained were verified as functional antibodies, such as high affinity antibodies, conformation-sensitive antibodies, flow cytometry applicable antibodies, and inhibitory antibodies, which indicates that this protocol is capable of producing membrane proteins with functionally correct conformations.
Another attractive benefit of this protocol is to allow the production of proteoliposomes that are assigned specific functions using modified lipids, such as biotinylated lipids, fluorescent lipids, or adjuvant lipids. Prepared proteoliposomes with specific functions are useful and applicable to a wide range of experiments. For example, adjuvant lipid-containing proteoliposomes, such as lipid A48 or monophosphoryl lipid A (MPLA)49, make convenient immunizing antigens, because they can be administered directly to immunize mice without emulsion. Adjuvant lipids effectively stimulate immune response in host animals, inducing antibodies against target membrane proteins (Figure 3A). Indeed, we have successfully induced flow cytometry applicable antibodies by immunizing mice with MPLA-containing proteoliposome31. Also, proteoliposomes prepared from biotinylated lipids are ideal probes for screening assays. We developed a high-throughput screening method to select anti-membrane protein antibodies using biotinylated proteoliposomes and AlphaScreen (BiLIA method) (Figure 3B)45. Sandwich ELISA is also able to be easily constructed using biotinylated proteoliposomes and streptavidin-coated plates.
Finally, there are two important caveats that should be addressed when using this method. First, formation of disulfide bonds may be insufficient due to the high concentrations of DTT in the translation buffer, which possibly affect the structure of some kinds of membrane proteins15. Although disulfide bonds are able to form after the reductant is removed during the purification process, they possibly form into different type rather than the natural ones. The other one is that membrane proteins are not glycosylated. The enzymes required for glycosylation are theoretically absent in the cell-free system because during the process when wheat germ extract is produced, biomembranes, including Golgi and ER, have been removed. Since the lack of disulfide bonds and glycosylation may cause different conformations, careful consideration and evaluation should be given to the experimental design, particularly when post-translational modifications are critical for the functional expression of proteins according to the experimental purposes.

Membrane proteins are one of the most important drug targets in diagnosis and therapeutics. Indeed, half of small compound drugs target are membrane proteins, such as G-protein-coupled receptors (GPCRs) and ion channels1. Over the years, researchers have been working on biochemical, biophysical, and structural studies of membrane proteins to elucidate their structure and function2,3. Development of monoclonal antibodies against membrane proteins is also performed actively in order to accelerate functional and structural studies and to develop therapeutic and diagnostic applications4,5,6,7,8,9. All these studies require a large amount of high quality membrane proteins10. For example, several milligrams of purified membrane proteins with natural conformation are needed for antibody development. A much larger quantity of highly purified membrane proteins are required for X-ray crystallography. However, mass production of membrane proteins remains a bottleneck in membrane protein research11. Membrane proteins have complicated structures with one or more transmembrane helices and play important roles in cell homeostasis. Heterologous overexpression of membrane proteins leads to multiple obstacles such as aggregation of membrane proteins that accumulate at high local concentrations or disturbance of cellular signal pathways. Even if the expression is successful, subsequent steps of sample preparation also face difficulty. For instance, preparation of proteoliposome, requires high-level skills and professional experiences in solubilization, purification, and stabilization of membrane proteins, and costs much effort and time as well12,13.
On the other hand, some advanced technologies have emerged in recent decades to produce proteins without the use of living cells14,15,16,17,18. Cell-free protein synthesis technology reconstitutes translation reaction in a test tube. Since there are no limitations that the cellular expression system has, cell-free systems have potential to synthesize a variety of proteins that are difficult to express or show toxicity in cells. Purified cell extract or reconstituted translational machinery is mixed with template mRNAs, amino acids, and energy sources, and recombinant proteins are synthesized in a short time. Regarding the membrane protein synthesis, some kinds of scaffolds composed of lipids or amphiphiles, such as liposomes, bicelles, nanodiscs, or copolymers are added to the cell-free reaction19,20,21,22,23,24. Synthesized membrane proteins interact with the scaffolds and can be stabilized in water. Cell-free synthesized membrane proteins are used widely in functional studies and antibody production25,26,27,28,29,30,31.
In this protocol, we describe an efficient cell-free method of proteoliposome production using wheat cell-free system and liposomes. Wheat cell-free protein synthesis system is a powerful in vitro translation system using extract from wheat germ15,32,33. Wheat germ contains a large amount of translational machinery and few translation inhibitors. The translational machinery in wheat, a member of eukaryotes, is suitable for translating eukaryotic proteins, and its translation efficiency is hardly affected by codon usage of the template mRNA. Using wheat cell-free system, we have synthesized a variety of proteins including protein kinases34,35, ubiquitin ligases36, transcription factors37, and membrane proteins with high success rates. For membrane protein production, we add lipid vesicle liposome into the translation mixture as scaffold19,38. Hydrophobic domains of membrane protein interact with lipid bilayer and are spontaneously integrated with liposome. Density gradient centrifugation is used to strictly separate proteoliposome from endogenous wheat proteins, even though a common centrifugation of the translation reaction mixture is sufficient for a simple purification of proteoliposome20. Many kinds of integral membrane proteins have been synthesized using wheat cell-free system and applied for various researches and developments25,38,39,40,41,42,43,44. Moreover, we developed the &#8220;bilayer-dialysis method&#8221; for large scale production45,46. In this method, cup-type dialysis device is immersed in the substrate feeding buffer, and two layers of translation reaction mixture and substrate feeding buffer are formed in the cup as shown in Figure 1. Continuous supply of substrates and removal of the byproduct can be efficiently conducted at both the top and the bottom of the reaction mixture for a long time, which leads to excellent translation efficacy (Figure 2A and Figure 2B)45.

<table><tbody><tr><td>&amp;times;3 SDS-PAGE sample buffer</td><td></td><td></td><td>Containing 10% 2-mercaptoethanol</td></tr><tr><td>5-20% gradient SDS-PAGE gel</td><td>ATTO</td><td>E-D520L</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td>Diluted ethanol by ultrapure water.</td></tr><tr><td>Agarose</td><td>Takara Bio</td><td></td><td></td></tr><tr><td>Ammonium acetate</td><td>Nakalai tesque</td><td>02406-95</td><td>As this reagent is deliquescent, dissolve all of it in water once opened and store it at -30&amp;deg;C.</td></tr><tr><td>Ampicillin Sodium</td><td>Nakalai tesque</td><td>02739-74</td><td></td></tr><tr><td>Asolectin Liposome, lyophilized</td><td>CellFree Sciences</td><td>CFS-PLE-ASL</td><td>A vial contains 10 mg of lyophilized liposomes.</td></tr><tr><td>BSA standard</td><td></td><td></td><td>1000 ng, 500 ng, 250 ng, 125 ng BSA / 10 &amp;micro;L &amp;times;1 SDS-PAGE sample buffer</td></tr><tr><td>CBB gel stain</td><td></td><td></td><td></td></tr><tr><td>cDNA clone of interest</td><td></td><td></td><td>Plasmid harboring cDNA clone or synthetic DNA fragment</td></tr><tr><td>Chloroform</td><td>Nakalai tesque</td><td>08402-84</td><td></td></tr><tr><td>Cooled incubator</td><td></td><td></td><td>Temperature ranging from 0 to 40 &amp;deg;C or wider.</td></tr><tr><td>Creatine kinase</td><td>Roche Diagnostics</td><td>04524977190</td><td></td></tr><tr><td>Dialysis cup (0.1 mL)</td><td>Thermo Fisher Scientific</td><td>69570</td><td>Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 0.1 mL</td></tr><tr><td>Dialysis cup (2 mL)</td><td>Thermo Fisher Scientific</td><td>88404</td><td>Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 2 mL</td></tr><tr><td>DNA ladder marker</td><td>Thermo Fisher Scientific</td><td>SM0311</td><td>GeneRuler 1 kb DNA Ladder</td></tr><tr><td>DpnI</td><td>Thermo Fisher Scientific</td><td>FD1703</td><td>FastDigest DpnI</td></tr><tr><td>E. coli strain JM109</td><td></td><td></td><td></td></tr><tr><td>Electrophoresis chamber</td><td>ATTO</td><td></td><td></td></tr><tr><td>Ethanol (99.5%)</td><td>Nakalai tesque</td><td>14713-95</td><td></td></tr><tr><td>Ethidium bromide</td><td></td><td></td><td></td></tr><tr><td>Evaporation flask, 100 mL</td><td></td><td></td><td></td></tr><tr><td>Gel imager</td><td></td><td></td><td></td></tr><tr><td>Gel scanner</td><td></td><td></td><td>We use document scanner and LED immuninator as a substitute.</td></tr><tr><td>LB broth</td><td></td><td></td><td></td></tr><tr><td>Lipids of interest</td><td>Avanti Polar Lipids</td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td>TOMY</td><td>MX-307</td><td></td></tr><tr><td>NTP mix</td><td>CellFree Sciences</td><td>CFS-TSC-NTP</td><td>Mixture of ATP, GTP, CTP, UTP, at 25 mM each</td></tr><tr><td>Nuclease-free 25 mL tube</td><td>IWAKI</td><td>362-025-MYP</td><td></td></tr><tr><td>Nucrease-free plastic tubes</td><td>Watson bio labs</td><td></td><td>Do not autoclave. Use them separately from other experiments.</td></tr><tr><td>Nucrease-free tips</td><td>Watson bio labs</td><td></td><td>Do not autoclave. Use them separately from other experiments.</td></tr><tr><td>PBS buffer</td><td></td><td></td><td></td></tr><tr><td>PCR purification kit</td><td>MACHEREY-NAGEL</td><td>740609</td><td>NucleoSpin Gel and PCR Clean-up</td></tr><tr><td>pEU-E01-MCS vector</td><td>CellFree Sciences</td><td>CFS-11</td><td></td></tr><tr><td>Phenol/chloroform/isoamyl alcohol (25:24:1)</td><td>Nippon Gene</td><td>311-90151</td><td></td></tr><tr><td>Plasmid prep Midi kit</td><td>MACHEREY-NAGEL</td><td>740410</td><td>NucleoBond Xtra Midi</td></tr><tr><td>Primer 1</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&amp;rsquo;-CCAAGATATCACTAGnnnnnnnnnnnnnnnnnnnnnnnn-3&amp;rsquo;&lt;br/&gt; Gene specific primer, forward. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&amp;hellip;. (20-30 bp) shows gene specific sequence.</td></tr><tr><td>Primer 2</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&#039;-CCATGGGACGTCGACnnnnnnnnnnnnnnnnnnnnnnnn-3&amp;rsquo;&lt;br/&gt; Gene specific primer, reverse. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&amp;hellip;. (20-30 bp) shows gene specific sequence.</td></tr><tr><td>Primer 3</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&#039;-GTCGACGTCCCATGGTTTTGTATAGAAT-3&#039;&lt;br/&gt; Forward primer for vector linearization. Underline works as overlap in seamless cloning.</td></tr><tr><td>Primer 4</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&#039;-CTAGTGATATCTTGGTGATGTAGATAGGTG-3&#039;&lt;br/&gt; Reverse primer for vector linearization. Underline works as overlap in seamless cloning.</td></tr><tr><td>Primer 5</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&amp;rsquo;-CAGTAAGCCAGATGCTACAC-3&amp;rsquo;&lt;br/&gt; Sequencing primer, forward</td></tr><tr><td>Primer 6</td><td>Thermo Fisher Scientific</td><td>Custom oligo synthesis</td><td>5&amp;rsquo;- CCTGCGCTGGGAAGATAAAC-3&amp;rsquo;&lt;br/&gt; Sequencing primer, reverse</td></tr><tr><td>Protein size marker</td><td>Bio-Rad</td><td>1610394</td><td>Precision Plus Protein Standard</td></tr><tr><td>Rotary evaporator</td><td></td><td></td><td></td></tr><tr><td>seamless cloning enzyme mixture</td><td>New England BioLabs</td><td>E2611L</td><td>Gibson Assembly Master Mix&lt;br/&gt; Other seamless cloning reagents are also avairable.</td></tr><tr><td>SP6 RNA Polymerase &amp;amp; RNase Inhibitor</td><td>CellFree Sciences</td><td>CFS-TSC-ENZ</td><td></td></tr><tr><td>Submarine Electrophoresis system</td><td></td><td></td><td></td></tr><tr><td>TAE buffer</td><td></td><td></td><td></td></tr><tr><td>Transcription Buffer LM</td><td>CellFree Sciences</td><td>CFS-TSC-5TB-LM</td><td></td></tr><tr><td>Translation buffer</td><td>CellFree Sciences</td><td>CFS-SUB-SGC</td><td>SUB-AMIX SGC (&amp;times;40) stock solution (S1, S2, S3, S4).&lt;br/&gt; Prepare &amp;times;1 translation buffer before use by mixing stock S1, S2, S3, S4 stock and ultrapure water.</td></tr><tr><td>Ultrapure water</td><td></td><td></td><td>We recommend to prepare ultrapure water by using ultrapure water production system every time you do experiment. Do not autoclave.&lt;br/&gt; We preparaed ultrapure water by using Milli-Q Reference and Elix10 system.&lt;br/&gt; Commercially available nuclease-free water (not DEPC-treated water) can be used as a substitute. Take care of contamination after open the bottle.</td></tr><tr><td>Ultrasonic homogenizer</td><td>Branson</td><td>SONIFIER model 450D-Advanced</td><td>Ultrasonic cleaner can be used as a substitute.</td></tr><tr><td>UV transilluminator</td><td></td><td></td><td></td></tr><tr><td>Vacuum desiccator</td><td></td><td></td><td></td></tr><tr><td>Wheat germ extract</td><td>CellFree Sciences</td><td>CFS-WGE-7240</td><td>WEPRO7240</td></tr></tbody></table>

cell-free production of proteoliposomes for functional analysis and antibody development targeting membrane proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Using this protocol, partially purified proteoliposomes can be obtained in a short time. Representative results are shown in Figure 2A. Twenty five GPCRs of Class A, B, and C were successfully synthesized using the bilayer-dialysis method (small scale) and partially purified by centrifugation and buffer wash. Although the amount of synthesized proteins varies according to the type of protein, 50 to 400 &#181;g of membrane proteins usually can be synthesized per reaction when large dialysis cups are used. Several milligrams of membrane proteins can be easily produced by increasing the number of reactions, due to the high scalability of wheat cell-free system. A pre-test using a small dialysis cup is sufficient to determine the production efficacy of the target protein in bilayer-dialysis method. According to the obtained productivity, the amount of the target protein to be produced using large dialysis cups can be estimated.
This protocol is suitable for expression of membrane proteins, particularly for those with multiple transmembrane helices. In most cases, membrane proteins with three or more transmembrane helices are easily incorporated into proteoliposomes after synthesis (Figure 2B), which makes a good productivity of proteoliposomes. Single-transmembrane-helix proteins are usually synthesized smoothly; however, they hardly integrate into liposomes due to the small hydrophobic region. Regarding proteins with two transmembrane helices, whether or not they are anchored to liposomes is dependent on the way their transmembrane helices are exposed.
Synthesized proteoliposomes are collected by simple centrifugation, and partially purified with a washing buffer, which greatly shortens the purification process of membrane proteins. Although both biological membranes and membrane proteins have been removed from wheat germ extracts beforehand, small amounts of wheat proteins are sometimes co-precipitated by binding to liposomes or membrane proteins synthesized (Figure 2A). Such protein contaminants are difficult to remove by simple centrifugation and buffer wash. When a highly purified membrane protein is required, it is necessary to solubilize the partially purified proteoliposomes with a surfactant and purify them by column chromatography.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61871/61871fig01.jpg" /> 
Figure 1: Scheme of cell-free proteoliposome production.&#160;SP6, SP6 promoter sequence; E01, E01 translation enhancer sequence; Ampr, ampicillin resistance gene; DTT, dithiothreitol. Electron micrograph shows immunogold labeling of biotinylated lipid containing liposome. Bar, 0.2 &#956;m. This electron micrograph image was from Figure 1D in Takeda et al., 201545. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61871/61871fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61871/61871fig02.jpg" /> 
Figure 2: Representative results of proteoliposome production by bilayer-dialysis method.&#160;(A) SDS-PAGE image of cell-free synthesized GPCRs. Twenty-five selected GPCRs were synthesized by the bilayer-dialysis method. Proteoliposomes were partially purified and applied to SDS-PAGE and CBB staining. Arrowheads indicate target GPCRs. (B) Comparison of membrane protein productions between different translation methods. Dopamine receptor D1 (DRD1) protein was synthesized by each method in the same ratio of wheat germ extract, liposomes, and mRNA, respectively. DRD1 proteoliposome was partially purified by centrifugation, and subjected to SDS-PAGE and CBB staining. (C) Immunogold labeling of DRD1-biotin/liposome complex. DRD1 was enzymatically biotinylated by BirA biotin ligase. Bar, 0.2 &#956;m. Blank arrowheads indicate DRD1-biotin on liposomes. This figure was modified from Figure 1 in Takeda et al., 201545. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61871/61871fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61871/61871fig03.jpg" /> 
Figure 3: Application of functional proteoliposomes.&#160;(A) Immunization of adjuvant lipid-containing proteoliposome. (B) Biotinylated liposome-based interaction assay (BiLIA). Interaction between membrane protein and anti-membrane protein antibody was detected by AlphaScreen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61871/61871fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins at JoVE.com

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

cell-free-production-proteoliposomes-for-functional-analysis-antibody

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.7K Views.  Ehime University. This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Video: Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Bioengineering

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Immunology and Infection

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Biology

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Summary

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Chapters in this video

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Antigenic Liposomes for Generation of Disease-specific Antibodies

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses