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

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 function^2,3. 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 applications

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.

1. 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 seamles....

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 a.......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
×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