Caspases,Paracaspases, and Metacaspases Methods and Protocols

9

the case for the expression of inactive caspases, the purified protein

is >95 % pure following IMAC chromatography (Fig. 2b). This

procedure allows for the purification of all apoptotic caspase cata-

lytic domains (Fig. 2c).

1. Transform BL21(DE3)pLysS competent cells with the appro-
   priate vector and spread the bacteria on LB agar plates with
   antibiotics. Incubate overnight at 37 °C. (DAY 1; see Note 5).


2. The following morning, inoculate 2 mL of 2× TY medium
   containing antibiotics with a small to medium colony of freshly
   transformed BL21(DE3)pLysS. Incubate in a 15 mL culture
   tube for ~8–10 h at 37 °C with vigorous shaking (250 rpm)
   (DAY 2).


3. In a 250 mL bacterial culture flask, dilute the primary culture
   100-fold into fresh 2× TY medium containing antibiotics and
   incubate as in step 1 for ~16 h (overnight). Prepare ~20 mL
   for each liter of final expression culture (see Note 6).


4. Set up 1 L baffled culture flasks (each containing 0.5 L of
   medium) by diluting the secondary culture 50-fold into 2× TY
   medium containing antibiotics. Incubate at 37 °C with vigor-
   ous shaking (250 rpm) until the optical density at 600 nm
   reaches between 0.5 and 0.7 (~2–4 h). Use sterile 2× TY
   medium as a spectrophotometer blank (DAY 3).


5. Decrease the temperature to 30 °C and induce expression by
   adding IPTG to a final concentration of 0.2 mM from a freshly
   made stock solution (48 mg/L of culture). Incubate at 30 °C
   with vigorous shaking (250 rpm) for ~5 h (see Note 7).


6. Once the expression period is over, transfer the culture to cen-
   trifuge bottles and collect the cells at 4 °C for 5 min at 3,900 × g.
   Discard the supernatant.


7. Resuspend the cell pellet in 10–15 mL of bacterial lysis buffer
   per liter of original culture volume (step 4). Purify immediately
   or store at −80 °C for up to 6 months in 50 mL polypropylene
   disposable screw cap tubes or an equivalent (see Note 8).


8. If frozen, thaw the bacterial suspension in tepid water. Do not
   let the suspension warm. Transfer the cell suspension into a
   50/100-mL plastic beaker. Keep on ice (DAY 4).


9. Using an ultrasonic homogenizer (large probe), break cells for
   2 min at 70 % power with a 50 % duty cycle (on for 0.5 s, then
   off for 0.5 s). Sonicate for 30–45 s/L of culture (see Note 9).


10. Transfer the lysate to centrifuge tubes and centrifuge at 4 °C
    for 30 min at 18,000 × g (12,000 rpm in a Sorvall SM-34 rotor
    or equivalent). The soluble fraction contains the caspase.


11. During centrifugation, pour 0.5–5 mL of Chelating Sepharose
    resin into an empty chromatography column (1.0 cm or less in

General Protocol
for Caspase Expression
in E. coli

General Caspase
Purification Protocol

Apoptotic Caspases Assays