Obtaining sufficient quantities of membrane proteins for downstream applications remains a significant technical challenge. This protocol enables the purification of several membrane transport proteins to homogeneity. A key advantage of this technique is that Saccharomyces cerevisiae is a time and cost effective eukaryotic expression system for membrane proteins that allows the optimization of many key experimental variables.
Begin by inoculating three transformed yeast colonies, in 50 milliliters of complete supplemental selective medium without histidine. Supplemented with yeast nitrogen base and 2%glucose for an overnight incubation at 190 rotations per minute and 30 degrees Celsius. Remove the culture the following day.
Use a spectrophotometer to determine the optical density at 600 nanometers or OD 600. And inoculate each of four two-liter flask containing 500 milliliters of culture medium to an OD 600 of 0.01. Then shake the cultures at 190 rotations per minute at 30 degrees Celsius for 30 hours to allow the cells to consume all of the glucose and to grow to a high density.
To induce borate transporter expression, add 125 milliliters of five x yeast peptone medium, supplemented with 10%galactose at 190 rotations per minute at 30 degrees Celsius for 16 hours. Then harvest the yeast cells by centrifugation and resuspend the pellet in 100 milliliters of cold water. Swirl in vortex to resuspend the yeast pellet and serially transfer the solution to do this with all bottles containing pellets.
To harvest the yeast membranes, first, add 11.25 milliliters of one molar Tris, 0.45 milliliters of 0.5 molar EDTA and 2.25 milliliters of 100 millimolar PMSF to the resuspended cells. Add water to a final volume of 225 milliliters and transfer the cells to a 450 milliliter metal bead beating canister. Top off the remaining volume with cold 0.5 millimeter glass beads and assemble the bead beading chamber with a rotor.
Immerse the chamber in an ice bath and perform six one-minute pulses, separated by two minute rest periods to prevent overheating of the lysate. After the last pulse, remove the filtration membrane from a plastic disposable bottle top filter screwed into a glass bottle and pour the contents of the bead beading chamber onto the assembly while applying vacuum. Then rinse with the two x bead wash buffer by adding to the chamber swirling and then adding to the beads.
Rinse the bead beading chamber with 225 milliliters of wash buffer and wash the beads with the contents of the chamber. Collect the lysate by centrifugation and decant the supernatant into polycarbonate bottles for ultracentrifugation for collection of the membranes. At the end of the ultracentrifugation discard the supernatant and weigh the bottles with the membrane pellets before resuspending the membranes in approximately 35 milliliters of membrane resuspension buffer.
Add the suspensions to a glass Douncer, then Dounce homogenize the membranes an aliquot the homogenate into a 50 milliliter conical tube for negative 80 degrees Celsius storage. Weigh the empty centrifuge bottles to determine the mass of the harvested membranes. For protein solubilization and purification, add a stir bar and 150 milligrams of DDM per gram of membrane to a beaker on ice and resuspend the thawed membranes to a final volume of 15 milliliters of membrane resuspension buffer, supplemented with one millimolar PMSF and 20 millimolar imidazoles per gram of membrane.
Add the membrane solution to the beaker for one hour of stirring in an ice bath. Followed by centrifugation to pellet the non-solubilized material. In a 4 degree celsius cold room, filter the supernatant through a five micrometer syringe filter and use a peristaltic pump set to a flow rate of one milliliter per minute to load the sample onto a one milliliter immobilized nickel affinity column Then, wash the column with 10 column volumes of wash buffer and dilute the protein in elution buffer.
Collecting the eluate in ten one milliliter fractions. Run the collected prepared gel fractions on a four to 20%Tris-Glycine SDS page gel along with solubilized lysate and wash fractions at room temperature, pulling the peak collected eluded fractions for concentration to a 500 micro liter or less volume in a 50 kilo dalton cutoff concentrator in a benchtop centrifuge at four degrees Celsius. Filter the concentrated protein through a 0.2 micrometer spin column filter and inject the filtrate onto a size-exclusion column equilibrated in a S-200 buffer.
After running the peak fractions on a second four to 20%Tris-Glycine SDS page gel, stain the gel and collect the pure peak fractions for concentration in a 50 kilo Dalton cutoff concentrator at four degrees Celsius as just demonstrated. Then measure the absorbance of the protein sample at a 280 nanometer wavelength to determine the concentration before storing the sample indefinitely at negative 80 degrees Celsius. To perform a glutaraldehyde cross-linking assay, first, add 0.5 milligrams per milliliter of thawed protein in three microliters of S-200 buffer to five microliters of S200 buffer.
Complete the negative control reaction with the addition of one microliter of 20%sodium dodecyl sulfate and one microliter of 1.5%glutaraldehyde. Complete the experimental samples with the addition of one micro liter of water and one micro liter of 1.5%glutaraldehyde. After 30 minutes at room temperature terminate the reaction with five microliters of 3x SDS page gel loading die containing an excess of Tris buffer to quench the glutaraldehyde and load all 15 micro liters of the solution onto a four to 20%Tris-Glycine SDS page gel.
Then, run the gel at 200 volts for 30 minutes before staining to determine the extent of the dimer cross-linking as evidenced by a band that runs at twice the size of the denatured monomer. Stain the gel by shaking on a slow orbital shaker with gel stain added to the gel. Here typical gels for the eluded fractions from nickel affinity chromatography show three partially purified proteins with the lysate fractions not demonstrating a significant band corresponding to the borate transporter as expected for proteins that do not over express well.
The ad millimolar wash lane in each gel shows minimal loss of the ten histidine tagged protein despite their relatively high emitters old concentration. While the bands corresponding to the borate transporters are readily apparent in the eluded fractions. Before S200 column injection, the proteins are insufficiently purified for many downstream applications as indicated by the extra bands in each sample.
After their injection into size-exclusion columns however, eluded fractions of a high purity can be observed. Crosslinking demonstrates that the purified homomeric transporters could have their assembly in solution readily assessed with the extent of cross-linking being dependent on the number of lysine residues in proximity to one another It is important to remember that once the cell harvesting begins, the protein must be kept cold at all times. Either on ice in a four degrees Celsius room or in a deli case.
Following this procedure, the membrane proteins can be further characterized by biophysical, biochemical or structural methods including X-ray crystallography or cryo-electron microscopy. By enabling the purification of milligram quantities of homogeneous protein, these techniques were used to determine the crystal structure of the Arabidopsis thaliana one transporter.
