The small scale plasma membrane isolation protocol and mGFPHis double tag, provide an economical, fast, reliable, and easy method to characterize membrane proteins in the eukaryotic model organism, Saccharomyces cerevisiae. The biggest advantage of this technology is the ease and speed with which membrane protein expression levels, the ATP's activities and the detergent is best suited for their purification can be determined. This technology is very user friendly.
However, one important point to remember is to harvest cells as OD 600 of 2, which ensures low mitochondrial contamination and the highest possible ATPase activities. Begin by pre-culturing a single yeast colony in 10 milliliters of YPD medium at 30 degrees Celsius for seven to eight hours at 200 rotations per minute. Use 10 milliliters of pre culture to inoculate 40 milliliters of fresh YPD medium.
Then incubate the cells at 30 degrees Celsius overnight at 200 rotations per minute until the cell density reaches an OD 600 of one to three. On the next day harvest 40 optical density units, or ODU, of logarithmic face cells by centrifuging at 4, 200 times G per five minutes at four degrees Celsius. Re-suspend and wash cells with 40 milliliters of ice cold sterile double distilled water.
Repeat the washing using one milliliter of ice cold water with centrifugalization between washing steps. Re-suspend the pellet in one milliliter of ice cold sterile water and transfer the cell suspension into a 1.5 milliliter micro centrifuge tube pre-cooled on ice. Harvest cells at 3, 300 times G for three minutes at four degrees Celsius.
Aspirate the supernatant and re-suspend the cell pellet in 500 microliters of homogenizing buffer freshly supplemented with one millimolar fenal methyl sulfa Neal fluoride, or PMSF. Store the cell suspension at minus 80 degrees Celsius until further use. Defrost the cells on ice for approximately one hour.
Then add ice cold 0.5 millimeter diameter silica beads to the 500 microliters of the cell suspension to reach a total volume of one milliliter. To break the cells, vortex the cell suspension at maximum shaking intensity for one minute, for six cycles, with a three minute cooling period on ice following each cycle. After vortexing, make a thin hole at the bottom of the tube with a heated scalpel blade and collect the broken cell homogenate into another ice cold 1.5 milliliter micro centrifuge tube with a low speed spin for 10 seconds.
Centrifuge the collected cell homogenate at 5, 156 times G for five minutes at four degrees Celsius to remove cell debris. Transfer 450 microliters of supernatant into an ice cold 1.5 milliliter micro centrifuge tube and add one milliliter of ice cold homogenizing buffer supplemented with one millimolar fresh PMSF. To harvest plasma membranes centrifuge the cell suspension at 17, 968 times G for one hour at four degrees Celsius.
Remove the supernatant and add 100 microliters of homogenizing buffer freshly supplemented with one millimolar PMSF. Then loosen the plasma membrane pellet by stirring with the 100 microliter pipette tip and re-suspend the pellet by repeat pipetting up and down. Measure the protein concentration of the plasma membrane preparation with a protein assay kit that is compatible with buffers containing the reducing agent and detergent.
Store the plasma membranes at minus 80 degrees Celsius, or keep them on ice for immediate use. Pour four to five milliliters of separating gel into the assembled gel apparatus up to two centimeters from the top. Carefully layer one to two milliliters of 0.1%SDS on top and allow the poly acrylamide gel to set for 60 minutes at room temperature.
After an hour remove the 0.1%SDS layer from the polymerized separating gel and rinse the gel with double distilled water to remove traces of SDS. Pour the stacking gel mix onto the separating gel. Place a comb into the stacking gel and remove any air bubbles from around the comb.
Then allow the stacking gel to set for 60 minutes at room temperature. Remove the comb and then rinse the gel slots with water. Put the gel into the gel tank and fill the gel tank to the top with running buffer.
Mix five to 10 microliters of plasma membrane samples with equal volumes of two times protein loading dye and immediately load the sample mixture into individual gel slots, submerged in running buffer. Load protein molecular weight markers in the range from 10 to 245 Kilodalton into a separate slot to enable the size estimation of individual protein fragments. Perform gel electrophoresis at 200 volts until the blue loading dye reaches the bottom of the gel.
Examine the gel for in-gel GFP fluorescence with a gel imaging system. Following the fluorescent imaging fix proteins in 10 to 20 milliliters of protein gel fixing solution by gentle agitation of the gel for 15 minutes at room temperature. Then rinse the twice for 10 minutes with 10 milliliters of double distilled water and place the gel in 10 milliliters of colloidal Kumasi stain solution with gentle shaking for one hour at room temperature to visualize protein bands.
For improved visualization of protein bands, de-stain the gel once or twice in 20 milliliters of double distilled water for one hour before recording images with the gel imaging system. A high frequency of transformation of Saccharomyces cerevisiae A D Delta Delta was achieved with the positive control plasmid PS2. No Eurocell Prototron transformants were obtained for the negative control.
About 50 URA cell prototroph CDR1-mGFPHis transformant were obtained after incubating transformed AD Delta Delta cells on CSM minus URA plates for three days. Petite transformant that cannot grow on YPG agar plates were eliminated. CDR1-mGHPHis samples with varying concentrations were quantified with in-gel fluorescence.
The mGFP fluorescence intensities were linear over the entire concentration range with minimal background fluorescence. Varying the cell density indicated that breaking 40 ODU of cells yielded the highest quality of plasma membranes with the highest CDR1 ATPase activity. However, the yield of the plasma membranes increased in proportion to increasing cell densities.
The ability of 31 detergents with various properties to solubilize CDR1-mGHPHis from crude plasma membranes of AD Delta Delta, CDR1-mGHPHis cells were tested with SDS page. Beta and alpha DDM And Fos-choline 13 were the best detergents to solubilize CDR1-mGHPHis. However, Fos-choline 13 cause partial hydrolysis of CDR1-mGHPHis.
Crude plasma membrane protein solubilized with 1%detergent was separated using a superos six increased 10 300 GL size exclusion column. Chromatograms of Maltosides and LMNG extracted proteins showed most CDR1-mGHPHis eluding as a nicely shaped Gaussian peak at 15.5 millimeters CDR1-mGHPHis solubilized with glucose containing detergents alluded as an aggregated or broad aggregated peak. The higher peaks for DDM indicated that a large portion of DMN G solubilized CDR1-mGHPHis may be denatured.
In the FSCC chromatograms for zwitterionic Fos-cholines and two non-ionic detergents only Digitonin and gave a similar chromatogram to DDM. Fos-choline 13 gave a symmetrical sharp shaped peak, but it alluded with a significantly lower elution volume than that for DDM. The optimized double tag also improve purification yield and helped determine the localization, trafficking, and thermal stability of membrane proteins.
The heterologous expression technology can also be used in high throughput drug screening, and the detailed characterization of many other membrane proteins.
