Our protocol addresses the reconstitution of a membrane protein within the lipid bilayer of a synthetic cell made using the inverted droplet emulsion method. The advantage of using the inverted droplet emulsion method is its ability to generate synthetic cells that provide a stable environment for the cell-free expression of membrane proteins. Our protocol offers a robust method for researchers in the field to synthesize membrane proteins inside synthetic cells using cell-free expression system.
My lab is working on reconstituting various ion channels in synthetic cells through the reconstitution of the action potential by a well-defined set of ion channels. To begin, blow a gentle stream of argon into a glass vial containing POPC lipids, while gently rotating the vial. When the lipids have dried as a film at the bottom of the vial, loosely cap it, then transfer it to a desiccator.
After one hour, add 0.5 milliliters of ultrapure deionized water to dissolve the lipid film. Vortex the solution for two minutes. Next, soak two filter supports in deionized water.
Place them on each of the internal membrane supports. Then place a 100-nanometer polycarbonate filter between the two internal membrane supports, held together by the extruder outer casing and retainer nut. Place the setup in the extruder stand to finish setting up the mini extrusion apparatus.
Next, load the lipid water mix into a one milliliter gas-tight syringe. Use swing arm clips to secure the syringe into one end of the mini extruder. Insert the second syringe into the other end of the mini extruder, making sure that it is fully depressed.
Now pass the lipid water mix from the original syringe into the empty one through the apparatus. After repeating the extrusion 11 times, transfer the SUVs to a 1.5 milliliter micro-centrifuge tube. To assemble the cell-free expression protocol, pipette Solutions 1 to 3, DNA plasmid encoding for soluble sfGFP or variants of glutamate receptor sfGFP.
murine RNAse, and SUV solution into a vial. Add ultrapure deionized water to bring the reaction volume to 20 microliters. Transfer the cell-free expression solution to a 96-well conical V-bottom plate.
Incubate the plate in a plate reader at 37 degrees Celsius for four to five hours. Set up the plate reader with a gain of 100, excitation of 485 nanometers, emission of 528 nanometers, a runtime of five hours, and an interval measurement of one read for every two minutes. Measure the GFP signal every two minutes to monitor the cell-free reaction in real time.
Soluble sfGFP had the highest expression among all three proteins. To begin, prepare the GUV outer buffer solution in a 1.5-milliliter micro-centrifuge tube. Mix together spermidine, ATP, GTP, CTP, UTP, creatinine phosphate, magnesium acetate, potassium glutamate.
HEPES, potassium hydroxide, folinic acid. glucose, and amino acid stock. Add ultra-purified deionized water to bring the final volume of the solution to one milliliter.
Next, in a fume hood, add 17.3 microliters of POPC stock solution to a 15-milliliter glass vial. To this, pipette 1.08 microliters of PEO-b-PBD copolymer. Carefully blow a gentle stream of argon gas into the glass vial while rotating the vial to evaporate the chloroform.
Then pipette 1.2 milliliters of light mineral oil into the glass vial. Vortex the lipid and oil at maximum speed for 10 to 20 seconds. Place the glass vial in an oven at around 50 degrees Celsius for 20 minutes before vortexing for an additional 10 to 20 seconds at maximum speed.
Next, pipette 270 microliters of the GUV outer solution into a 1.5-milliliter micro-centrifuge tube. To this, pipette 15 microliters each of five molar sodium chloride and 4.5 molar potassium chloride. Gently pipette 300 microliters of lipid and oil mixture on top of the GUV outer solution.
After incubation, assemble a CFE reaction by pipetting Solutions 1 to 3, DNA plasmid encoding for soluble sfGFP or variants of glutamate receptor sfGFP, murine, RNAse, and one molar sucrose, into a vial. Add ultrapure deionized water to bring the reaction volume to 20 microliters. Add 600 microliters of the lipid oil mixture to the micro-centrifuge tube containing the 20-microliter reaction mixture.
Pipette up and down vigorously for one minute to emulsify the reaction. Gently layer the inner emulsion on top of the oil layer in the tube. Centrifuge for 10 minutes at 2, 000 g at four degrees Celsius.
Next, carefully pipette out the excess oil and outer solution from the micro-centrifuge tube. Gently mix the GOV pellet in the remaining solution to resuspend it. Transfer the GOV solution to a clean 96-well clear flat-bottom plate for incubation.
Transfer the sealed plate into a plate reader to incubate at 37 degrees Celsius for five to six hours. After incubation, place the plate on an inverted confocal microscope. Focus on a region of interest containing the GUVs.
Then capture the images at an excitation wavelength of 488 nanometers. Save the images in the TIFF format. Open the images in an image processing software.
Click on the Brightness/Contrast setting panel to open it. Then adjust the brightness and contrast to make the fluorescent proteins visible. The Wild Type-Glutamate Receptor cell-free expression reactions encapsulated in GUV demonstrated excellent expression and membrane localization.
PRSP-Glutamate Receptor was prone to aggregation and punctate formation. The soluble sfGFP was expressed in GUVS and stayed in the GUV lumen.
