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 ...

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>&#215;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&#176;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 &#181;L &#215;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 &#176;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&#8217;-CCAAGATATCACTAGnnnnnnnnnnnnnnnnnnnnnnnn-3&#8217; 
			Gene specific primer, forward. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&#8230;. (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&#39;-CCATGGGACGTCGACnnnnnnnnnnnnnnnnnnnnnnnn-3&#8217; 
			Gene specific primer, reverse. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn&#8230;. (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&#39;-GTCGACGTCCCATGGTTTTGTATAGAAT-3&#39; 
			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&#39;-CTAGTGATATCTTGGTGATGTAGATAGGTG-3&#39; 
			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&#8217;-CAGTAAGCCAGATGCTACAC-3&#8217; 
			Sequencing primer, forward</td>
		</tr>
		<tr>
			<td>Primer 6</td>
			<td>Thermo Fisher Scientific</td>
			<td>Custom oligo synthesis</td>
			<td>5&#8217;- CCTGCGCTGGGAAGATAAAC-3&#8217; 
			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 
			Other seamless cloning reagents are also avairable.</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase &#38; 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 (&#215;40) stock solution (S1, S2, S3, S4). 
			Prepare &#215;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. 
			We preparaed ultrapure water by using Milli-Q Reference and Elix10 system. 
			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 c...

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 ...

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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

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

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 ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...