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.
All the membrane proteins are important targets in drug discovery. The largest scale production of membrane proteins remains a bottleneck to conducting biochemical, biophysical, and structural membrane protein studies. This method can be used to efficiently synthesize membrane proteins in-vitro and to prepare purified proteoliposomes in a short time with a high success rate.
After adding the appropriate reagents, including the cell-free expression plasmid to generate a transcription reaction mixture, mix the tube contents by gentle inversion or tapping and perform a quick spin in a microcentrifuge. After the spin, incubate the transcription reaction mixture for six hours at 37 degrees Celsius followed by mixing and a spin down step. To prepare the translation buffer, dilute the stock solution by fresh ultrapure water.
For small scale translation, use 100 microliter dialysis cups washed with ultrapure water to remove the glycerol from the dialysis membrane. Add one milliliter of ultrapure water to a new 1.5 milliliter tube and insert a small scale dialysis cup into the tube. Then add 500 microliters of ultrapure water to the cup for a 30-minute incubation at room temperature.
To prepare liposomes, transfer a lipid solution containing 50 milligrams of lipids to an evaporation flask. Place the flask in a rotary evaporator to evaporate the solvent. When a thin, evenly spread layer of lipid has been generated on the surface of the flask bottom, transfer the flask to a vacuum desiccator under negative pressure overnight to remove the solvent completely.
The next morning, remove the flasks from the desiccator and check the thin film of lipids. Add one milliliter of translation buffer to the flask and rotate the flask to spread the buffer over the thin lipid film. After five minutes, sonicate the flask changing the angle occasionally to allow the solution to contact the entire lipid film surface.
Stop sonication when the lipid is completely stripped from the flask and the liposome suspension becomes uniform. Then transfer the liposome suspension to a new 1.5 milliliter tube. Before performing an in-vitro protein translation, float a tube of wheat germ extract from minus 80 degrees storage on water at room temperature for a few minutes to quickly thaw the material.
Upon thawing the extract, immediately mix the tube contents by gentle inversion. Then spin down and place the tube on ice. Add the wheat germ extract mRNA and liposomes to generate a translation reaction mixture.
Mix the tube contents by gentle inversion or tapping and perform a quick spin in a microcentrifuge. To conduct a small scale in-vitro protein translation, remove the water from the small scale dialysis cup and tube and add one milliliter of translation buffer to the tube and 300 microliters to the cup. Take 60 microliters of translation reaction mixture.
Insert the pipette tip into the translation buffer in the dialysis cup. Then inject the mixture slowly and gently. The reaction mixture with higher density spontaneously sinks to the bottom of the cup and forms a layer.
Cover the cup with a lid to prevent evaporation. To conduct a large scale translation, add 22 milliliters of translation buffer to a 25 milliliter tube and place a two milliliter dialysis cup into the tube. Add two milliliters of translation buffer to the dialysis cup.
Take 500 microliters of the translation reaction mixture, slowly and gently inject the mixture into the translation buffer in the cup. Incubate the reactions for 24 hours at 15 degrees Celsius. The next day thoroughly mix the reaction in the dialysis cup by pipetting and transfer the crude proteoliposome suspensions to new tubes.
To purify the proteoliposomes, sediment the proteoliposome in the crude suspensions by centrification. Then wash the proteoliposome by resuspending in the appropriate volume of ice cold PBS buffer three times. After washing, thoroughly resuspend the proteoliposome pellets in a small volume of PBS and measure the suspension volume by using a micropipette.
Then add PBS to adjust the volume of suspension. Transfer each suspension into a new micro tube. To set up an SDS page analysis, add 70 microliters of water and 40 microliters of three time concentrated SDS page sample buffer to 10 microliters of each proteoliposome suspension.
Set up a five to 20%gradient SDS page gel in an electrophoresis chamber. Load the gel with two microliters of the protein size marker three, six, and 12 microliters of each proteoliposome sample and 10 microliters of bovine serum albumin standards. After loading samples, run the electrophoresis followed by staining the gel with Coomassie Brilliant Blue dye.
Finally, decolorize the gel in hot water with gentle shaking and scan the gel image. Using this protocol as demonstrated, a variety of membrane proteins with three or more transmembrane helices can be easily produced as proteoliposomes in a short time. In this bilayer dialysis method, a continuous supply of substrates and the removal of the byproduct can be efficiently conducted at both the top and bottom of the reaction mixture over a long period of time, leading to an excellent translation efficacy.
The bilayer dialysis method results in a four to 10 times increase in the productivity compared to using the bilayer or dialysis methods alone. Collection of the synthesized proteoliposomes by centrification and their partially purification with a washing buffer greatly shortens the purification process of the membrane proteins. This cell-free system is easy to manipulate and highly scalable.
It can also be used to produce memory proteins that are difficult to express using other methods. This method is extremely helpful for investigations and experiments that use or target membrane proteins such as antimembrane protein, antibody production, SPR, Elisa, and AlphaScreen.
