Caspases are cysteine proteases that cleave substrates during apoptosis and non-apoptotic processes. This protocol describes an in vitro cleavage assay to identify putative substrates of the initiator caspase Dronc from Drosophila. If followed carefully, this protocol provides a reliable procedure for high throughput screening of putative substrates of the caspase Dronc, or any other caspase.
While this protocol has been designed for in vitro cleavage assays using the Drosophila caspase Dronc, it can easily be adapted for caspases from other organisms, including mammals. It is important that the purification of the recombinant caspases and the in vitro cleavage assays are performed on the same day because these caspase preparations lose enzymatic activity quickly. Demonstrating the procedure will be Dr.Prathibha Yarikipati, a post-doctoral fellow from my laboratory.
To begin, remove the frozen tubes with the pelleted cultures from minus 80 degrees Celsius storage and keep them on ice for 10 minutes to soften the pellet. After 10 minutes, add 0.6 milliliters of bacterial cell lysis buffer supplemented with freshly added protease inhibitors, 10 milligrams per milliliter of lysozyme, and 50 units per milliliter of Benzonase, into the pellet containing tubes using a pipette controller with a one milliliter serological pipette. With the same pipette tip, dissolve the pellet by pipetting up and down until a clear, pale yellow solution, without any pellet particles, is visible and incubate for 30 minutes on ice.
Transfer lysates into appropriate centrifuge tubes and centrifuge them at 17, 000 G for 40 minutes at 4 degrees Celsius. Transfer the supernatants into a 1.5 milliliter microcentrifuge tube, which is the crude extract containing the caspases, and keep the tubes on ice. Add 0.2 milliliters of the 50%slurry of Ni-nitriloacetic acid agarose to each tube of the caspase extracts, and rotate the tubes on an end-on-end rotator for one hour at 4 degrees Celsius.
After one hour, add the extract with the Ni-nitriloacetic acid agarose to one milliliter polypropylene columns with a tip intact, placed in a rack, and let it stand for five minutes to allow Ni-nitriloacetic acid agarose to settle. After five minutes, remove the cap of the columns, and let the supernatant flow out by gravity flow. Then carefully add one milliliter of the wash buffer to the columns without disturbing the packed resin of Ni-nitriloacetic acid agarose, and wash it through gravity flow.
For elution of the caspases, place 1.5 milliliter microcentrifuge tubes under the collecting nozzle of the columns, after complete draining of the wash buffer. Then, add 0.5 milliliters of the elution buffer to each column and collect the eluant in the 1.5 milliliter microcentrifuge tubes. Keep the eluants on ice and measure the concentration of proteins by Bradford assay.
Depending on the expression level of the putative substrate, use 1 to 10 microliters of the rabbit reticulocyte lysate programmed with the protein of interest in the cleavage assay and keep it on ice. Add 10 microgram of purified caspase protein generated previously, and bring the total reaction volume to 50 microliters with caspase assay buffer. Incubate the reactions in a water bath at 30 degrees Celsius for three hours, ensuring to include the appropriate controls in the cleavage assay.
After three hours, stop the reactions by transferring the tubes on ice, and add one volume of LDS sample buffer containing 50 millimolar DTT. Incubate the samples at 75 degrees Celsius in a heat block for 10 minutes. Quickly spin, mix by flicking, load 24 microliters per sample and run the SDS page or store the samples at minus 20 degrees Celsius.
Four different recombinant caspases were induced, purified, and analyzed by SDS-PAGE, immunoblotted, and the blot was probed with an antihistidine antibody. The unprocessed 6x-Histidine Dronc runs at a relative molecular weight of 55 kilodalton and the unprocessed 6x-His DRICE has a molecular weight of 35 kilodalton. Auto processing of the caspases is visible by the appearance of bands of smaller molecular weight.
For cleaved Dronc, it is a band of 40 kilodalton. For cleaved Drice, it is a band of 23 kilodalton. After the in vitro cleavage reaction, the proteins were separated by SDS-PAGE, immunoblotted, and the blots were incubated with an anti-Myc antibody to detect amino terminally Myc-tagged Drice C211A.
The results demonstrated that the bacterially-produced and purified 6x-histidine Dronc wild-type preparation has enzymatic activity. This is visible by the presence of the smaller band of cleaved Drice compared to uncleaved proDrice. The in vitro cleavage reaction that uses both recombinant caspase and substrate was analyzed by SDS-PAGE and immunoblot.
For analysis of the cleavage reaction, the anti cleaved Drice antibody was used in this immunoblot. The results demonstrated that the bacterially-produced and purified 6x-His Dronc preparation has enzymatic activity. This is visible by the presence of the smaller band of cleaved Drice compared to the uncleaved proDrice.
Please remember that recombinant caspases lose enzymatic activity fast. They cannot be stored, not even at minus 80 degrees Celsius. The in vitro cleavage assays have to be performed on the same day.
Positive hits in the in vitro cleavage assay should be validated in vivo. This can be done in cells or in genetic model organisms such Drosophila or C.elegans.
