This article describes a method for synthesizing membrane protein antigens using a bacterial cell-free system. The process involves the use of plasmids, synthetic phospholipids, and a dual-compartment dialysis device to facilitate protein synthesis and assembly into lipid nanodiscs.
Take a bacterial cell-free system that lacks intact cellular structures but retains the molecular components necessary for in vitro transcription and translation.
Introduce plasmids encoding the membrane protein antigen and the scaffold protein, along with synthetic phospholipids and polymers.
Supplement the mixture with ATP, GTP, and an amino acid mix to initiate protein synthesis.
Simultaneously, prepare a feed solution enriched with the amino acid mix.
Load the mixture into the inner chamber and the feed solution into the outer chamber of a dual-compartment dialysis device.
During incubation, the substrates diffuse from the outer to the inner chamber through the semi-permeable membrane, facilitating plasmid transcription and protein translation.
The scaffold protein assembles the phospholipids and polymers into lipid nanodiscs and facilitates the folding and incorporation of MPA into these structures.
By-products generated during the reaction diffuse into the outer chamber, maintaining optimal conditions for nanodisc-stabilized MPA formation.
Prepare MOMP-tNLPs by employing a cell-free method.
Defrost the reconstitution buffer from the cell-free protein expression kit two hours before setting the reaction, and add EDTA-free protease inhibitor to the buffer. Use a kit designed to run five one-milliliter reactions. For each one-milliliter reaction, add 525 microliters of reconstitution buffer to the E.coli lysate bottle and roll it to dissolve.
Add 250 microliters of the reconstitution buffer to the reaction bottle containing the additive and dissolve by rolling. Next, add 8.1 milliliters of reconstitution buffer to the reaction feed bottle, and roll it to dissolve. To a bottle of amino acid mixture, add three milliliters of reconstitution buffer to dissolve it.
To another bottle of methionine, add 1.8 milliliters of reconstitution buffer, dissolve it, and store it on ice. Add 225 microliters of the reconstituted mixture, 270 microliters of the reconstituted amino acid mixture without methionine, and 30 microliters of reconstituted methionine to the E.coli lysate bottle. Further, add 400 microliters of the DMPC/telodendrimer mixture 15 micrograms of MOMP plasmid, and 0.6 micrograms of delta-49 ApoA1 to the mixture and mix it.
Aliquot 20 microliters of the total solution in a 1.5-milliliter tube For a GFP-expressing control reaction. Prepare a feed solution by adding 2.65 milliliters of the reconstituted amino acid mixture without methionine and 300 microliters of reconstituted methionine. Transfer one milliliter of the reaction solution to the inner reaction chamber in the cell-free reaction kit and seal it.
Fill the outer chamber of the reaction vessel with 10 milliliters of the feed solution and close it. Add 0.5 microliters of the GFP control plasmid to the 20-microliter aliquots of the reaction mixture. Put the reaction in a shaker at 300 rotations per minute for 18 hours at 30 degrees Celsius.
Monitor the reaction under a UV light after 15 minutes for fluorescence due to GFP synthesis.
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