Ontluikende Gist Protein extractie en zuivering voor de Studie van de functie, interacties, en post-translationele modificaties

The overall goal of this procedure is to extract yeast proteins while preserving native structure function interactions and post-translational modifications. This is accomplished by first growing cells to induce expression of the protein of interest. Next, the harvested yeast cells are homogenized in a suitable buffer to extract the proteins.

Then the proteins of interest are purified from the clarified whole cell extract using an affinity of resin. The final step of the procedure is to elude the purified proteins from the affinity resin for further analysis or downstream applications. Ultimately, results can be obtained that show protein purification modifications and interactions based on western blot analysis.

Demonstrating the procedure will be Eva's Ky a William and Mary graduate who spent the last couple of years as an undergraduate researcher in my lab. The main advantage of bead beading over other existing methods is the rapid processing and lysis of multiple samples under conditions that preserve protein function interactions and post-translational modifications. To begin transform cells of a gal positive yeast strain with a plasmid encoding a galactose inducible six, his tagged protein of choice inoculate transformants in five milliliters of appropriate selective medium such as SD uracil, containing 2%sucrose and incubate at 30 degrees Celsius overnight with rotation the following day dilute the overnight culture in selective medium with 2%sucrose to a final volume of 33 milliliters so that the optical density at 600 nanometers measure 0.3, grow in a shaking incubator at 30 degrees Celsius and 150 RPM.

When the culture has reached an OD 600 of 0.8 to 1.5, induce expression of the desired protein by adding 17 milliliters of three XYEP with 6%galactose for a final concentration of one XYEP with 2%galactose placed in a shaking incubator at 30 degrees Celsius for an additional five to six hours. After measuring the OD 600 of the induced culture centrifuge about 150 to 200 OD 600 units of cells for five minutes at 5, 000 RPM at four degrees Celsius, then use one milliliter of ice cold one XPBS with one x protease inhibitor cocktail to re suspend the cell palate and transfer it to a two milliliters screw cap tube centrifuge the cells for one minute at 15, 000 RPM and four degrees Celsius. After decanting the supernatant snap freeze the cell pellet in liquid nitrogen to the frozen cell pellet.

Add 200 microliters of acid washed glass beads and 500 microliters of ice cold lysis buffer. Keeping the tubes on ice at all times. Use a pipette tip with the end cutoff to briefly pipette up and down at four degrees Celsius.

Place the tubes of cells into the bead mill balance lock and run the machine as per the manufacturer's instructions for 20 seconds at 5.5 meters per second. Then place the tubes on slushy ice for one minute. Repeat a total of six times to clear the extracted proteins centrifuge for 15 minutes at 15, 000 RPM at four degrees Celsius.

Then to prepare a sample of the whole cell extract or WCE for western blot and WCE corresponding to two OD 600 units of cells to 800 microliters of 20%trichloroacetic acid or TCA vortex to mix. To purify a sample of clear WCE at 50 to 100 microliters of affinity resin to a new micro centrifuge tube wash and equilibrate the resin by adding one milliliter of wash buffer and invert top over bottom until the resin is resuspended. After spinning for one minute at 5, 000 RPM and four degrees Celsius, aspirate the supernatant to bind protein for affinity purification at 100 to 200 microliters of cleared lysate to the washed beads and use lysis buffer to bring the total volume to one milliliter.

Incubate with mutation or rocking at four degrees Celsius for two to five hours. Use liquid nitrogen to snap freeze aliquots of the remaining clear WCE and store at minus 80 degrees Celsius until further use, while the lysate beet solution is incubating for the WCE Western blot sample on ice. After spinning for two and a half minutes at 15, 000 RPM at four degrees Celsius, decant the supernatant taking care to retain the pellet, add 800 microliters of 2%TCA to the pellet and vortex the tube before repeating the two and a half minute spin.

After carefully decanting the supernatant, add 100 microliters of TCA sample buffer and vortex to dissolve the pellet before incubating the sample. In a 100 degree Celsius heat block for two to five minutes for takes a second time to fully dissolve any remnants of the pellet, which can be difficult to dissolve and store at minus 80 degrees Celsius until ready to use. Once the lysate bead sample is incubated, after spinning at 5, 000 RPM and four degrees Celsius for one minute, use wash buffer to wash the resin five times to elute the proteins.

Add 150 microliters of elution buffer to the resin mu tate at four degrees Celsius for five minutes. Spin for one minute at 5, 000 RPM and four degrees Celsius and save the supernatant in a new tube. Finally, to prepare a sample for western blot at 25 microliters of two x lithium docal sulfate sample buffer with two microliters of BME to 25 microliters of UD protein sample.

As shown in this figure, a wide range of proteins can be reproducibly extracted from yeast cells. Discrete bands over a range of molecular weights are indicative of high quality protein extracts. The quality of extracts can be confirmed by western blotting for specific epitope tagged proteins.

Here is a representative result for the galactose overexpressed V five tagged Sumo Ligase is one which migrates at about 120 kilodaltons. Generally, partially degraded proteins would run as multiple fragments below the expected weight, but here only full length protein is observed. Additionally, high molecular weight adds of a given protein may indicate post-translational modifications.

For example, here you can see lactose over expressed sumo related S one delta four 40 and full length SIS one. As demonstrated in this Western blot modifications can also be preserved on endogenously expressed S one. This figure shows that two nuclear enriched proteins SLX five and CS one delta four 40 were successfully purified from our ws.

Notably, a six hiss tagged protein can be affinity purified from W'S, both under native and denaturing conditions. In contrast, SLX 5G ST, and CS one delta four 40 ha lack a six hiss epitope, but under native conditions still interact with the metal affinity resin in an ole sensitive manner. We propose that CIS one and to a lesser extent SLX five are able to bind the metal affinity resin via their metal coordinating ring domain.

While to our knowledge, this particular phenomenon has not yet been reported, a similar situation has been described in which chole toxin B sub unit binds Nicola affinity resin in a manner mediated by its natural histamine residues. This observation suggests that proteins in the WCER, at least in part, natively folded for the study of protein function. It is important to show that protein complexes remain intact in whole cell extracts.

A representative data revealed that CS one delta four 40 SLX five, and P GK one a cytosolic protein that serves as a loading control were present in WS cis one. Delta four 40 was purified from these extracts based on its inherent ability to bind to metal affinity resins. In addition, when SLX five and SIS one were co-expressed in yeast cells, SLX five levels in the EITs were increased raising the possibility that SLX five may interact with SIS one.

After watching this video, you should have a good understanding of how to grow and process yeast cells in order to extract, purify and visualize yeast proteins under native conditions.