The protocol is highly efficient and versatile. It can be applied to almost any membrane protein, and the whole production procedure is completed within a day. The synthesized membrane proteins can be stabilized already co-translationally by supplied ligands.
Detergent contacts are completely avoided, and the proteins insert and fold directly in provided and tailored membrane environments. Structural approaches, functional characterization, and antibody generation are prime areas of application. The technique is moreover straightforward in analyzing lipid affects on membrane protein folding and function.
On day three of culture, inoculate 10 liters of sterile 37 degree Celsius YPTG medium in a 15-liter stirred-tank reactor with 100 milliliters of pre-culture. And cultivate the bacteria at 37 degrees Celsius with 500 revolutions per minute in high aeration. To prevent excessive foaming, add sterile anti-foam.
When the cells reach the mid-log phase, add 300 milliliters of ethanol to the culture and incubate the cells at 42 degrees Celsius with 500 rotations per minute in high aeration for another 45 minutes. At the end of the incubation, harvest the cells by centrifugation, and use a spatula and a pipette to resuspend the cells completely in 300 milliliters of S30A buffer. Centrifuge the resuspended bacteria two times, washing the pellet with 300 milliliters of fresh S30A per wash.
After the second wash, resuspend the cells in a 1.1 volume of S30B buffer. Load the suspension into a pre-cooled French-press pressure cell and disrupt cells at 1, 000 pounds per square inch gauge one time. To remove any unstable components and to release the mRNA from the ribosomes, adjust the lysate to 0.4 molar sodium chloride and incubate the sample for 45 minutes in a 42-degree Celsius water bath.
To assemble the membrane scaffold protein nanodiscs, mix the MSP1E3D1 solution with the lipid solution and add DPC to a final concentration of 0.1%Adjust the reaction to the appropriate final volume according to the table and incubate the reactions for one hour at 21 degrees Celsius under gentle agitation. At the end of the incubation, pre-wet the membrane of a dialysis cassette with disc-formation buffer and use a syringe to load the assembly mixture into the cassette. Next dialyze the reaction against three five-liter volumes of disc-formation buffer for 10 to 20 hours at room temperature with stirring before transferring the mixture into disc formation-buffer equilibrated centrifugal filter units with a 10 kilodalton molecular weight cutoff.
Then concentrate the reaction to at least 300 micromolar by centrifugation using a UV-Vis reader to measure the concentration. To prepare a three-milliliter continuous exchange cell-free reaction, equilibrate the membrane of a three-milliliter dialysis cassette in 100 millimolar tris-acetate. And use a syringe to transfer the reaction mixture into the cassette.
Using a syringe, remove excess air from the reaction mixture compartment and place the cassette into the dialysis chamber. Fill the chamber with 60 milliliters of feeding mixture and place the lid on the chamber. After fixing the lid into place, incubate the chamber for 12 to 16 hours at 30 degrees Celsius in 200 revolutions per minute.
To prepare a 60-microliter cell exchange cell-free reaction setup for magnesium ion screening, add 60 microliters of our freshly prepared 3X Master Mix to the reaction mixture and 825 microliters to the feeding mixture. And fill the feeding mixture with water. After vortexing, add 825 microliters of feeding mixture to each of three wells of a 24-well microplate.
Cut regenerated cellulose dialysis tubes with a 10 to 14 kilodalton molecular weight cutoff into approximately 25-by-20 millimeter-sized pieces, and store the pieces in water supplemented with 0.05%sodium azide. Before mini-cell exchange cell-free container assembly, use a tissue to remove any excess water from the dialysis membranes, and place one membrane piece on each container. Fix the membranes with a polytetrafluoroethylene ring and transfer the mini container into one well of feeding mixture.
Add the high-molecular components to the reaction mixture, as indicated in the table, and mix by pipetting. Add 60 microliters of reaction mixture to the reaction container, taking care to avoid air bubbles. And wrap two eight-by-10 centimeter sheets of a sealing thermoplastic film around the 24-well plate.
Fix the lid of the well plate in place with tape and place the plate at 30 degrees Celsius in 200 revolutions per minute with agitation for 12 to 16 hours. The next morning, use a pipette tip to pierce through the dialysis membrane at the mini-cell exchange cell-free container and aspirate the reaction mixture. Transfer the reaction mixture into new centrifuge tubes and centrifuge to remove precipitates.
Then transfer the supernatant into a new tube. Here a representative impact of fine-tuning reaction compounds on the final yield or quality of synthesized membrane proteins is shown. In this analysis, the overall synthesis and nanodisc solubilization of the T-beta-one adrenergic receptor was similar under all of the analyzed membrane compositions.
In contrast, a much higher variation was detected in the quality of the synthesized G protein-coupled receptor, with the lowest activity obtained with the DMPC and POPC lipids, and the highest observed when DOPG and POPG were applied. Taken together, these results indicate that the charge of the lipid head group as well as the flexibility of the fatty acid chain are important modulators for the folding and activity of this cell membrane protein. The final nanodisc concentration can also be an important factor for membrane protein quality.
For example, if the nanodisc concentration is screened within a range of 3.75 to 60 micromolar, a complete solubilization of the G protein-coupled receptor is obtained at approximately 30-micromolar nanodiscs, giving a one-to-three membrane protein-to-nanodisc ratio. In contrast, a complete solubilization of proteorhodopsin can be achieved with approximately 10-micromolar nanodiscs, giving a 10-to-one proteorhodopsin-to-nanodisc ratio. Accurate pipetting and high-quality components are certainly mandatory.
Critical steps for lysate preparation are harvesting the cells in mid-log phase and applying the high-salt step. The technique drastically reduces complexity of membrane protein synthesis. Formerly challenging targets are efficiently synthesized, hence allowing to work on molecular structures of so far unexplored pharmaceutically relevant proteins.
