Caspases,Paracaspases, and Metacaspases Methods and Protocols

79

cells are plated onto LB/agar containing the appropriate

antibiotic (ampicillin, kanamycin, and/or chloramphenicol)

for selection of transformed bacterial colonies. Bacterial colo-

nies are grown on LB/agar petri dishes overnight at 37 °C.

2. Single colonies are inoculated into 5 ml LB (with antibiotic)
   and cultured overnight (O/N) with shaking at 37 °C.


3. 5 ml O/N culture is subcultured 1:20 into 200 ml LB (with
   antibiotic) and incubated at 30 °C for 3 h with shaking.


4. Add IPTG to a fi nal concentration 1 mM, and incubate a fur-
   ther 4 h with shaking at 30 °C.


5. Pellet bacterial cells by centrifugation at 6,000 × g for 15 min at
   4 °C. Store the bacterial pellet at −20 °C overnight.


6. Resuspend pellet in 10 ml PBS and lyse bacteria by sonication
   (5 × 20 s pulses)


7. Add Triton X-100 to fi nal concentration of 0.1 %, and incu-
   bate 30 min at 4 °C.


8. Clarify by centrifugation at 15,000 × g for 30 min at 4 °C and
   transfer cleared supernatant to a new tube. At this stage, the
   lysate can be used in a caspase-2 activity assay (Subheading 3.2 )
   to determine the activity level of recombinant protein.


9. Recombinant caspase-2 protein is now ready to purify on glu-
   tathione Sepharose beads (Caspase-2-GST) or Ni-NTA beads
   (Caspase-2-His 6 ) ( see Notes 5 and 6 ).


10. Wash glutathione Sepharose beads or Ni-NTA beads in PBS.


11. Add 50 μl bead volume to the cleared lysate and incubate at
    4 °C for 1 h with gentle rotation.


12. Transfer beads to a chromatography column (e.g., Poly-prep
    chromatography column from Bio-Rad) and wash beads three
    times with 10 ml of PBS.


13. Elute recombinant caspase-2 protein in 5 bead volumes of elu-
    tion buffer in fi ve separate fractions and quantitate protein
    concentration using a standard BCA or Bradford assay.


14. Purifi ed protein can also be checked by resolving protein on
    SDS-PAGE and proteins then stained with Coomassie Brilliant
    Blue.

Caspase activity assays are typically carried out over a time course

(i.e., every 10–15 min over a 1–2 h time course). The spectropho-

tometer should be equipped with a regulated temperature cham-

ber that can accommodate 96-well plates. Alternatively the release

of AFC, AMC fl uorescent tags can be measured following different

time points over a fi xed period of incubation at 37 °C. The addi-

tion of a caspase-2 inhibitor (z-VDVAD-FMK or Ac-VDVAD-

CHO) or pan-caspase inhibitors (z-VAD-FMK or Q-VD-OPh)

3.2 Measuring
Caspase-2 Activity

Assays for Studying Caspase-2