増殖に基​​づく決定とタンパク質分解に対する遺伝的要件の生化学的確認で<em>サッカロマイセス·セレビシエ</em

The overall goal of the following experiment is to rapidly determine the genetic requirements for degradation of an unstable protein in Croce Visier. This is achieved by fusing a reporter enzyme required for growth under specific selective conditions to an unstable protein of interest. Next wild type or mutant cells both expressing the fusion protein are cultured on selective medium agar plates.

The plating assay provides a convenient indicator of protein stabilization on the basis of yeast cell growth in order to biochemically confirmed differences in protein abundance while type in mutant cells harboring the fusion protein are lysed via a rapid inefficient protein extraction method for Western blot analysis. The results show that the abundance of an unstable protein is increased in cells with a mutation in a gene required for degradation. The converse is true for degradation competent wild type strains.

The main advantage of this technique over existing methods like pulse chase and Cycl heide chase analyses is that is less labor intensive and time consuming, allowing for the rapid and high throughput determination of genetic requirements for protein degradation. Though this method can provide insights into the degradation requirements for a range of unstable proteins. It can also be used to identify regulators of other aspects of gene expressions such as transcription and translation.

Demonstrating the procedures will be Sheldon Watts and Justin Crowder. Students in my laboratory Prior to the assay transform a plasmid encoding a fusion protein consisting of an unstable protein and a metabolic reporter enzyme into a mutant yeast strain. Suspected of having genetic defects pertaining to regulation of protein degradation as a control, also transform the same plasmid into a wild type yeast strain.

Inoculate the transformants in five milliliters of minimal medium that selects for cells, harboring the plasmid molecules. Place the test tube into an inclined rotator and incubate overnight at 30 degrees Celsius on the next day. Measure the optical density of the overnight cultures, then equalize the cell concentration for all cells by diluting each culture to an OD 600 of 0.2 with fresh media and sterile individual 1.5 milliliter centrifuge tubes.

To begin the main assay, set up a sixfold dilution series matrix by first transferring 200 microliters of each pre-diluted strain, starting from an OD 600 of 0.2 into designated wells in the first column of a sterile 96 well plate in preparation for subsequent dilutions, use a multi-channel pipette to add 125 microliters of sterile water into the empty wells in columns two, three, and four. Next, transfer 25 microliters of yeast from column one into column two, pipette up and down a few times to mix the cells evenly and proceed to transfer 25 microliters of cells from column two into column three. Repeat the serial dilution steps until the wells in column four are well mixed as a visual aid for cell spotting.

Print out a grid template and tape it on the inside of a Petri dish lid, beginning with the least concentrated column in the serial dilution matrix. Transfer four microliters of cells from each column. On the 96 well plate onto a control minimal medium agar plate that selects for intracellular plasmid maintenance Spot another four microliters of cells from each column onto a separate, minimal medium agar plate that selects for both intracellular plasmid.

Maintenance and expression of the unstable fusion protein. Allow both plates to dry on the bench for five to 10 minutes. Seal the lids with param to prevent dehydration and incubate the cells at 30 degrees Celsius for two to six days.

When colonies from the fastest growing culture first become visible at the most dilute spot on a given plate, remove the plate from the incubator and document the extent of growth. By taking photographs, it may be necessary to take photographs of different plates on different days or of the same plate on multiple days. In the case of different yeast strains that exhibit a wide range of growth rates, so biochemically confirmed that a gene identified in the growth assay regulates protein abundance begin by transforming wild type and mutant yeast strains with a plasmid encoding the aforementioned fusion protein, inoculate the transformants in five milliliters of minimal medium that selects for intracellular plasmid maintenance and incubate the tubes overnight in a 30 degrees Celsius inclined rotator.

On the next day, measure the optical density at 600 of each overnight culture. Once all strains have reached an OD 600 of 0.4 or above, dilute all cultures back down to 0.2 with fresh medium such that the final volume for each strain is 10 milliliters. Incubate the cells on a 30 degrees Celsius platform shaker until all cultures reach a mid log phase growth OD 600, between 0.8 and 1.2.

Harvest a cells by transferring 2.5 total OD units of cells into a fresh 15 milliliter conical tube. Centrifuge the cells at room temperature for five minutes and remove the S supernatant. Then add one milliliter of distilled water vortex or pipette up and down to resuspend the palate and transfer the cell suspension to a fresh 1.5 milliliter tube.

Isolate the cells with the second centrifugation for 30 seconds At room temperature, remove the snat and resuspend the cells in 100 microliters of distilled water. Add 100 microliters of 0.2 molar sodium hydroxide to the cell suspension, pipette up and down a few times to mix and incubate the samples for five minutes at room temperature To avoid premature cellular lysis. Do not vortex the cells.

Isolate the cells again with a third centrifugation for 30 seconds. At room temperature, remove the supernatants from the cells and lys the cells by resus suspending the pellet in 50 to 100 microliters of one x lamby sample buffer denature all proteins by incubating the suspension at 95 degrees Celsius for five minutes, and then cool the whole cell lysate on ice for five minutes. For assaying the abundance of aggregation prone proteins such as proteins with several transmembrane segments empirically determine the optimal denaturing incubation, temperature, and time by starting at 70 degrees Celsius for 10 minutes instead of 95 degrees Celsius for five minutes.

Finally, isolate the soluble fraction by centrifuging the lysate for one minute at room temperature, transfer the snat into a fresh tube and discard the insoluble pellet fraction. The soluble fraction can now be loaded directly onto an SDS page, gel or stored at negative 20 degrees Celsius in setting up a yeast growth-based protein degradation assay. An unstable triple fusion protein is fused with an additional metabolic enzyme that compliments yeast strain background lacking the same metabolic gene.

When the fusion protein is expressed in yeast, it is prone to a barren translocation events at the endoplasmic reticulum membrane. This aberrant translocation triggers ubiquitin mediated degradation of the entire protein, including the metabolic enzyme, thus preventing cell proliferation. However, in yeast strains lacking the ubiquitin ligase, the fusion protein is protected from degradation, even upon average translocation allowing cells to proliferate.

Therefore, in the presence of ubiquitin ligase, there is a correspondence between protein degradation occurring inside the cells and failure of yeast cells to proliferate on metabolite depleted agar plates. On the other hand, in the context of a defective or absent ubiquitin ligase cell proliferation indicates that the fusion protein was protected from degradation to reduce background or spurious growth and increase the stringency of this assay. Competitive inhibitors of the metabolic enzyme may be added to the growth medium to confirm results observed in the yeast growth assay.

Direct evidence of fusion protein degradation can also be ascertained with a western blot. In the presence or absence of ubiquitin ligase, the fusion protein will be degraded or spared from destruction respectively. It is important to remember that these procedures report most directly on the steady state abundance of the protein of interest.

After these procedures have been conducted, pulse or cycloheximide chase analyses may be performed in order to directly analyze protein degradation kinetics and the presence of mutations found to increase study state protein levels.