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

Wei Zhou; Hiroyuki Takeda

doi:10.3791/61871

JoVE Journal > Biochemistry

Biochimica

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

Published: September 22, 2020

doi:

10.3791/61871

Wei Zhou¹, Hiroyuki Takeda²

¹Graduate School of Medicine,Ehime University, ²Proteo-Science Center,Ehime University

Summary

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.

Abstract

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 “bilayer-dialysis method” 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.

Introduction

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 “bilayer-dialysis method” 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.

Protocol

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.

Prepare insert DNA fragment.
1. 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.
2. Add 1/25 volume of DpnI restriction enzyme to the PCR product to remove template plasmid DNA. Incubate for 30 min at 37 °C.
3. Use a PCR purification kit to purify the PCR product and adjust concentration at 20–50 ng/µL.
Linearize pEU-E01-MCS vector.
1. Conduct inverse PCR using pEU-E01-MCS, primer 3, and primer 4.
2. Add 1/25 volume of DpnI restriction enzyme to the inverse PCR product. Incubate for 30 min at 37 °C.
3. Use a PCR purification kit to purify the PCR product as per the manufacturer’s recommendation. Adjust the concentration at 20–50 ng/µL.
Mix 2 µL of insert DNA fragment, 2 µL of linearized vector, and 4 µL of 2x seamless cloning enzyme mixture.
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.
Confirm the sequence of the expression plasmid constructed using primer 5 and primer 6 from 5’ and 3’ side of MCS in pEU plasmid, respectively.
Amplify and purify the expression plasmids.
1. Culture the plasmid-transformed E. coli strain JM109 in 150 mL of LB-ampicillin medium at 37 ˚C and 125 strokes per minute shaking overnight.
2. Extract and purify the plasmids using commercially available plasmid prep midi kit. Dissolve plasmids in 500 µL of TE buffer.
  CAUTION: Do not use a mini prep kit for plasmid extraction. It does not provide sufficient quality and quantity of plasmid.
3. Add 500 µL of phenol/chloroform/isoamyl alcohol (25:24:1). 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.
  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.
4. Add 500 µL 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.
5. Add 2.5 volume of ethanol and 1/8 volume of 7.5 M ammonium acetate, and store at -30 °C for 1 h.
6. Centrifuge at 17,800 x g at 4 °C for 10 min. Wash the pellet with 500 µL of 70% ethanol. Remove the supernatant carefully and leave the pellet for 5 min to dry up.
7. Dissolve the expression plasmids in 100 µL of ultrapure water completely. Measure the concentration of plasmids with absorbance at 260 nm. Adjust the concentration to 1 mg/mL.

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.

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.
Prepare transcription reaction mix by mixing 115.2 µL of ultrapure water, 40 µL of Transcription Buffer LM, 20 µL of NTP mix, 2.4 µL of 80 U/µL RNase inhibitor, 2.4 µL of 80 U/µL SP6 polymerase, and 20 µL of 1 mg/mL pEU expression plasmids. Mix the reagents gently by inverting. Perform a quick spin.
Incubate the transcription reaction at 37 °C for 6 h.
Mix the reaction gently by inverting and quickly spin down. Use it immediately for translation, otherwise freeze and store at -80 °C.
Confirm the transcription product by electrophoresis.
1. 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.
2. Take 1 µL of transcription reaction and mix with 3 µL of water and 4 µL of 2x loading dye.
  NOTE: Denaturing of RNA is not required.
3. Load 4 µL of the mixture and 2 µL of DNA ladder marker to the agarose TAE gel.
4. Electrophorese at 100 V for 20 min.
5. 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.

3. Preparation of materials for translation

Prepare the translation buffer.
1. 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.
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 µL each) in 0.2 mL 8-strip PCR tubes. Freeze the tubes in liquid nitrogen, and store at -80 °C.
CAUTION: Do not re-freeze creatine kinase solution after thawing.
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.
1. 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.
2. Incubate for more than 30 min at room temperature.

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).

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.
1. Open the vial containing 10 mg of lyophilized Asolectin liposomes (see the Table of Materials) and add 200 µL of translation buffer (section 3.1) to the bottom of the vial. Seal the vial and incubate for 10 min.
2. Mix vigorously by putting the vial on the vortex-mixer for 1 min.
3. Insert the vial into a 50 mL tube. Spin down the tube by centrifuging at 500 x g for 1 min.
4. 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 °C.
Prepare liposomes by hydrating a thin lipid film.
1. 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.
2. Transfer the lipid solution containing 50 mg of lipid(s) to an evaporation flask.
3. 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.
4. Put the flask in a vacuum desiccator and leave under negative pressure overnight to remove the solvent completely.
5. 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.
6. 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.
7. 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 °C.

5. In vitro translation

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 °C. It withstands several freeze/thaw cycles.
Thaw 20 mg/mL creatine kinase stock solution. Mix 5 µL of stock solution and 45 µL of translation buffer to prepare 2 mg/mL creatine kinase working solution.
CAUTION: Refreezing creatine kinase is not recommended.
Thaw liposomes or mRNAs when required.
Conduct a small-scale protein translation.
1. Remove water from both the tube and the dialysis cup (0.1 mL), as prepared in step 3.3.2.
2. Inject 1 mL and 300 µL 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 50–100 µL of buffer to the tube.
3. Prepare translation reaction mixture by mixing 15.6 µL of translation buffer, 2.4 µL of 2 mg/mL creatine kinase, 12 µL of 50 mg/mL liposomes, 15 µL of wheat germ extract, and 15 µL of mRNA. Mix gently by inverting the tubes, and spin down.
4. Aspirate 60 µL of the translation reaction mixture using a 200 µL pipette.
5. 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.
Conduct a large-scale translation (Figure 1).
1. 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.
2. Prepare a translation reaction mixture by mixing 130 µL of translation buffer, 20 µL of 2 mg/mL creatine kinase, 100 µL of 50 mg/mL liposome, 125 µL of wheat germ extract, and 125 µL of mRNA. Mix gently by pipetting.
3. Aspirate all the translation reaction mixture (500 µL) using a 1,000 µL 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.
Incubate the reactions at 15 °C for 24 h.
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.

6. Purification of proteoliposomes

Centrifuge the tube containing crude proteoliposome suspension at 17,800 x g at 4 °C for 10 min.
Remove the supernatant. Suspend the proteoliposome pellet in PBS (small scale: 1 mL, large scale: 10 mL) by pipetting.
Repeat the centrifugation and washing of proteoliposomes for another two circles.
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 µL (small scale) or 500 µL (large scale). Transfer the suspension to a new 1.5 mL tube.
Transfer 10 µL 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 °C.

7. SDS-PAGE and CBB staining

Add 70 µL of water and 40 µL of 3x SDS-PAGE sample buffer to 10 µL 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.
Set a 5%–20% gradient SDS-PAGE gel in an electrophoresis chamber. Load 3 µL, 6 µL, 12 µL of proteoliposome samples, 2 µL of protein size marker, and BSA standard series as well.
Electrophorese at 52 mA, 400 V for 30 min.
Stain the gel with CBB dye for 1 h. Decolorize in hot water and scan the gel image.
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.

Representative Results

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 µg 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.

Figure 1: Scheme of cell-free proteoliposome production. SP6, SP6 promoter sequence; E01, E01 translation enhancer sequence; Amp^r, ampicillin resistance gene; DTT, dithiothreitol. Electron micrograph shows immunogold labeling of biotinylated lipid containing liposome. Bar, 0.2 μm. This electron micrograph image was from Figure 1D in Takeda et al., 2015⁴⁵. Please click here to view a larger version of this figure.

Figure 2: Representative results of proteoliposome production by bilayer-dialysis method. (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 μm. Blank arrowheads indicate DRD1-biotin on liposomes. This figure was modified from Figure 1 in Takeda et al., 2015⁴⁵. Please click here to view a larger version of this figure.

Figure 3: Application of functional proteoliposomes. (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. Please click here to view a larger version of this figure.

Discussion

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 4–10 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.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

Riferimenti

Santos, R., et al. A comprehensive map of molecular drug targets. Nature Reviews. Drug Discovery. 16 (1), 19-34 (2017).
Gusach, A., et al. Beyond structure: emerging approaches to study GPCR dynamics. Current Opinion in Structural Biology. 63, 18-25 (2020).
Congreve, M., de Graaf, C., Swain, N. A., Tate, C. G. Impact of GPCR Structures on Drug Discovery. Cell. 181 (1), 81-91 (2020).
Wilkinson, T. C. I. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels. Biochemical Society Transactions. 44 (3), 831-837 (2016).
Hino, T., Iwata, S., Murata, T. Generation of functional antibodies for mammalian membrane protein crystallography. Current Opinion in Structural Biology. 23 (4), 563-568 (2013).
Webb, D. R., Handel, T. M., Kretz-Rommel, A., Stevens, R. C. Opportunities for functional selectivity in GPCR antibodies. Biochemical Pharmacology. 85 (2), 147-152 (2013).
Douthwaite, J. A., Finch, D. K., Mustelin, T., Wilkinson, T. C. I. Development of therapeutic antibodies to G-protein coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases. Pharmacology and Therapeutics. 169, 113-123 (2016).
Hashimoto, Y., Yagi, K., Kondoh, M. Current progress in a second-generation claudin binder, anti-claudin antibody, for clinical applications. Drug Discovery Today. 21 (10), 1711-1718 (2016).
Hutchings, C. J., Colussi, P., Clark, T. G. Ion channels as therapeutic antibody targets. mAbs. 11 (2), 265-296 (2019).
Errey, J. C., Fiez-Vandal, C. Production of membrane proteins in industry: The example of GPCRs. Protein Expression and Purification. 169, 105569 (2020).
Pandey, A., Shin, K., Patterson, R. E., Liu, X. Q., Rainey, J. K. Current strategies for protein production and purification enabling membrane protein structural biology. Biochemistry and Cell Biology. 94 (6), 507-527 (2016).
Wiseman, D. N., et al. Expression and purification of recombinant G protein-coupled receptors: a review. Protein Expression and Purification. 167, 105524 (2020).
Jeffery, C. J. Expression, Solubilization, and Purification of Bacterial Membrane Proteins. Current Protocols in Protein Science. 83 (1), 1-15 (2016).
Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y., Alakhov, Y. B. A continuous cell-free translation system capable of producing polypeptides in high yield. Science. 242 (4882), 1162-1164 (1988).
Takai, K., Sawasaki, T., Endo, Y. Practical cell-free protein synthesis system using purified wheat embryos. Nature Protocols. 5 (2), 227-238 (2010).
Endo, Y., Sawasaki, T. Cell-free expression systems for eukaryotic protein production. Current Opinion in Biotechnology. 17 (4), 373-380 (2006).
Shimizu, Y., Kuruma, Y., Ying, B. W., Umekage, S., Ueda, T. Cell-free translation systems for protein engineering. The FEBS Journal. 273 (18), 4133-4140 (2006).
Klammt, C., et al. Cell-free expression as an emerging technique for the large scale production of integral membrane protein. The FEBS Journal. 273 (18), 4141-4153 (2006).
Nozawa, A., et al. A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant and Cell Physiology. 48 (12), 1815-1820 (2007).
Nozawa, A., et al. Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system. BMC Biotechnology. 11 (1), 35-45 (2011).
Henrich, E., Hein, C., Dotsch, V., Bernhard, F. Membrane protein production in Escherichia coli cell-free lysates. FEBS Letters. 589 (15), 1713-1722 (2015).
Henrich, E., Peetz, O., et al. Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology. eLife. 6, 243 (2017).
Shelby, M. L., He, W., Dang, A. T., Kuhl, T. L., Coleman, M. A. cell-free co-translational approaches for producing mammalian receptors: expanding the cell-free expression toolbox using nanolipoproteins. Frontiers in Pharmacology. 10, 744 (2019).
Jacobs, M. L., Boyd, M. A., Kamat, N. P. Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression. Proceedings of the National Academy of Sciences of the United States of America. 116 (10), 4031-4036 (2019).
Sackin, H., Nanazashvili, M., Makino, S. I. Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA. Channels. 9 (4), 196-199 (2015).
Zemella, A., Richter, T., Thoring, L., Kubick, S. A combined cell-free protein synthesis and fluorescence-based approach to investigate GPCR binding properties. Methods in Molecular Biology. 1947 (10), 57-77 (2019).
Vaish, A., Guo, S., Murray, R. M., Grandsard, P. J., Chen, Q. On-chip membrane protein cell-free expression enables development of a direct binding assay: A curious case of potassium channel KcsA-Kv1.3. Analytical Biochemistry. 556, 70-77 (2018).
Suzuki, Y., et al. Functional G-Protein-Coupled Receptor (GPCR) synthesis: the pharmacological analysis of Human Histamine H1 Receptor (HRH1) synthesized by a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. Frontiers in Pharmacology. 9, 38 (2018).
Cortes, S., Barette, C., Beroud, R., De Waard, M., Schaack, B. Functional characterization of cell-free expressed Kv1.3 channel using a voltage-sensitive fluorescent dye. Protein Expression and Purification. 145, 94-99 (2018).
Woznicka-Misaila, A., Juillan-Binard, C., Baud, D., Pebay-Peyroula, E., Ravaud, S. Cell-free production, purification and characterization of human mitochondrial ADP/ATP carriers. Protein Expression and Purification. 144, 46-54 (2018).
Hashimoto, Y., et al. Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5. Scientific Reports. 8 (1), 8383 (2018).
Sawasaki, T., et al. A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Letters. 514 (1), 102-105 (2002).
Goshima, N., et al. Human protein factory for converting the transcriptome into an in vitro-expressed proteome. Nature Methods. 5 (12), 1011-1017 (2008).
Nemoto, K., Takemori, N., Seki, M., Shinozaki, K., Sawasaki, T. Members of the plant CRK superfamily are capable of trans- and autophosphorylation of tyrosine residues. The Journal of Biological Chemistry. 290 (27), 16665-16677 (2015).
Takeda, H., et al. Comparative analysis of human src-family kinase substrate specificity in vitro. Journal of Proteome Research. 9 (11), 5982-5993 (2010).
Takahashi, H., et al. Establishment of a wheat cell-free synthesized protein array containing 250 human and mouse E3 ubiquitin ligases to identify novel interaction between E3 ligases and substrate proteins. PLoS One. 11 (6), 0156718 (2016).
Nozawa, A., et al. Construction of a protein library of Arabidopsis transcription factors using a wheat cell-free protein production system and its application for DNA binding analysis. Bioscience, Biotechnology, and Biochemistry. 73 (7), 1661-1664 (2009).
Goren, M. A., Nozawa, A., Makino, S. I., Wrobel, R. L., Fox, B. G. Cell-free translation of integral membrane proteins into unilamelar liposomes. Methods in Enzymology. 463, 647-673 (2009).
Nozawa, A., et al. Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system. BMC Biotechnology. 11 (1), 35 (2011).
Renauld, S., et al. Functional reconstitution of cell-free synthesized purified Kv channels. Biochimica Et Biophysica Acta. Biomembranes. 1859 (12), 2373-2380 (2017).
Liu, S., et al. Efficiency and Safety of CRAC Inhibitors in Human Rheumatoid Arthritis Xenograft Models. Journal of Immunology. 199 (5), 1584-1595 (2017).
Jarecki, B. W., Makino, S. I., Beebe, E. T., Fox, B. G., Chanda, B. Function of shaker potassium channels produced by cell-free translation upon injection into xenopus oocytes. Scientific Reports. 3, 1-7 (2013).
David, G., et al. Phosphorylation and alternative translation on wheat germ cell-free protein synthesis of the DHBV large envelope protein. Frontiers in Molecular Biosciences. 6, 138 (2019).
Jirasko, V., et al. Proton-detected solid-state NMR of the cell-free synthesized α-helical transmembrane protein NS4B from hepatitis C virus. Chembiochem: A European Journal of Chemical Biology. 21 (10), 1453-1460 (2020).
Takeda, H., et al. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Scientific Reports. 5, 11333 (2015).
Zhou, W., Takeda, H. Production of immunizing antigen proteoliposome using cell-free protein synthesis system. Methods in Molecular Biology. 1868, 49-67 (2018).
Hutchings, C. J., Koglin, M., Marshall, F. H. Therapeutic antibodies directed at G protein-coupled receptors. mAbs. 2 (6), 594-606 (2010).
Raetz, C. R. H., et al. Discovery of new biosynthetic pathways: the lipid A story. Journal of Lipid Research. 50, 103-108 (2009).
Baldridge, J. R., Crane, R. T. Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods. 19 (1), 103-107 (1999).

Play Video

PDF

DOI

DOWNLOAD MATERIALS LIST

Citazione di questo articolo

Zhou, W., Takeda, H. Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins. J. Vis. Exp. (163), e61871, doi:10.3791/61871 (2020).