Class 1 histone deacetylases like RpdA are discussed as potential novel targets for the treatment of fungal infections. However, purified enzyme activities are required for the further characterization. The main advantage of this protocol is the rapid and sufficient separation of native TAP-tagged complexes for activity determination in one single step.
HDAC complexes derived from this protocol may be used for efficacy screening of novel fungal-specific deactylase inhibitors. This method provides the basis for the extraction and purification of TAP-tagged fungal proteins, which might be used as starting point for the establishment of protocols for other enzymes and strains. To begin this procedure, add 10 milliliters of CSS to each flask of prepared conidia.
Tightly close each flask with the provided screw cap and shake vigorously. After this, use a sterile inoculation loop to completely scrape off any remaining conidia. Pass the conidia through 40-micrometer cell strainers placed onto a centrifuge tube and collect the suspension from five of the flasks into one tube.
Then centrifuge the samples to combine and dilute the samples as outlined in the text protocol. First place cheesecloth into a funnel on top of a flask. Filter the prepared mycelia through the cloth and wash briefly with deionized water.
Remove as much moisture as possible from the mycelia by squeezing the cheesecloth between just hands and then between paper towels. After this, transfer the dried mycelia as flat sheets into a plastic beaker with a screw lid. Using liquid nitrogen, flash-freeze the mycelia and store it at minus 80 degrees Celsius prior to lyophilization.
Then lyophilize the mycelia overnight. The next day, stop the freeze-drying process when the temperature of the mycelia remains constant. Remove the beakers and immediately seal them with the provided screw caps.
First add 1.5 grams of mycelia and a grinding ball into the grinding jar of a ball mill. Grind the mycelial powder at 25 hertz for 30 seconds and transfer the mycelial powder to a 15-milliliter centrifuge tube. Tilt the tube to allow subsequent mixing of the mycelia with the buffer, then add six milliliters of ice-cold extraction buffer B250, including 1X protease inhibitor cocktail, per gram of mycelial powder and blend with a small spatula until complete homogenization of the crude extract is achieved;keep the tube on ice for five minutes.
Next place the tube and a balance tube into a centrifuge and spin at 40, 000 times g and at four degrees Celsius for at least 20 minutes. During the centrifugation, set out a 10-milliliter disposable chromatography column to begin equilibrating the IgG resin. Pipette 300 microliters of well-resuspended IgG resin into the column.
Fill the column to 10 milliliters with B250 and let the buffer flow through by gravity. Add one milliliter of B250 including 1X protease inhibitor cocktail and let it flow through, then plug the bottom of the column. After the centrifugation, remove 10 microliters of the supernatant for SDS-PAGE analysis.
Place the sample into a 1.5-milliliter tube containing 40 microliters of water and 12.5 microliters of 5X LSB. Using a serological pipette, carefully remove the supernatant and transfer it onto the column containing the equilibrated IgG beads. Close the column by tightly securing the provided end cap.
To begin, incubate the chromatography column on a rotary mixer at 10 rpm and at four degrees Celsius for two to four hours. After this, remove the cap and open the column at the bottom to collect the flow-through. To wash the column, use a pipetter to add one milliliter of washing buffer 250 to the column cap to remove any trapped beads and transfer this suspension in one flush onto the settled resin to resuspend the beads.
Then fill the column up to the top with washing buffer 250 and close it using a stack cap connected to a peristaltic pump. Start the peristaltic pump and adjust the pump to a flow rate of approximately one to five milliliters per minute, repeat this washing process for a total of four washes, then repeat this washing process three times using TEV equilibration buffer. Close the chromatography column at the bottom.
Resuspend the IgG beads in one milliliter of TEV cleavage buffer and add 20 microliters of 50X protease inhibitor cocktail as well as 10 microliters of TEV. Next cap the column and incubate on a rotary mixer at 10 rpm and at four degrees Celsius overnight to elute the protein complexes bound via the tagged HDAC. The next day, open the column and collect the eluate in a two-milliliter centrifuge tube.
Use 0.7 milliliters of TEV cleavage buffer to remove the beads from the cap and to rinse the wall of the column. Place the two-milliliter centrifuge tube into a 50-milliliter centrifuge tube and then place the column onto the open two-milliliter tube. Transfer this whole assembly onto a tabletop centrifuge and spin at 300 times g for two minutes to obtain the TEV eluate.
In this study, a single-step enrichment of a TAP-tagged Class 1 HDAC from the filamentous fungus Aspergillus nidulans is performed for the assessment of in vitro deactylase activity. A typical outcome of this clearly illustrates the efficacy of the first affinity step, which is even further increased when performing the tandem purification. Most of the prominent proteins present in the protein extract and the flow-through, however, already are depleted in the TEV eluate.
An immunoblot shows strong signals migrating at approximately 120 kilodaltons corresponding to CBP-tagged full-length RpdA in the TEV eluate, the calmodulin flow-through, and eluate fractions. A representative deactylase activity assay with the specific HDAC inhibitor Trichostatin A is shown here. The sensitivity of the activity confirms that the measured values are due to RpdA and not caused by unspecific protease activity.
This is important as it indicates that TEV protease, which is present at rather high concentration, does not interfere with the HDAC activity assay. Interestingly, HDAC activity is significantly reduced after the second affinity purification step, CE, when compared to the TEV eluate. In order to allow efficient protein extraction, ensure that mycelia are ground to fine powder.
This is particularly critical when no machine is available and mortar and pestle are used for grinding. The addition of the second affinity step to the protocol results in fractions pure enough for protein identification but done on mass spectrometry. When working with liquid nitrogen, make sure to wear safety goggles and protective gloves to avoid personal injury.
