12
series of fractions containing full-length caspase-8-YFP (~84 kDa)
and main contaminants eluting prior to the pool of caspase. DEAE
anion exchange chromatography (Fig. 3b), in addition to
concentrating the caspase, allows for the removal of more impuri-
ties. Following refolding, the typical emission spectrum of YFP is
recovered (Fig. 3c), along with enzymatic activity (Fig. 3d).
The following protocols are valid for bacterial cultures of
1–2 L and can be easily scaled up.The expression protocol is based on the general procedure
described in Subheading 3.1.1 with the modification that caspase-8
expression induction is performed using 0.4 mM IPTG at 37 °C
for 5 h (step 5, Subheading 3.1.1.1) (DAYS 1–3).- 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). - Using an ultrasonic homogenizer (large probe), break cells for
2 min at 70 % power with 50 % duty cycle (on for 0.5 s, then
off for 0.5 s). Keep on ice. - 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). Discard the supernatant. The insoluble pellet
contains the caspase. - Suspend the inclusion body pellet in 10–20 mL of guanidine
buffer. Transfer to a small plastic beaker and stir overnight at
room temperature to solubilize the proteins. Keep everything
at room temperature from this step forward (see Note 16). - The next morning, centrifuge the solubilized proteins for
30 min at 18,000 × g (12,000 rpm in a Sorvall SM-34 rotor or
equivalent) to eliminate remaining insoluble debris. The super-
natant contains the solubilized caspase (DAY 5). - Prepare the Chelating Sepharose as described in
Subheading 3.1.1.2, step 4, but equilibrate the column with 5
bed volumes of guanidine buffer. Allow 2 mL of resin to purify
from 1 L of bacterial expression. - Resuspend the prepared resin with the supernatant from step
5 in a 15–50 mL tube. Incubate with gentle shaking for ~2 h. - Recover the resin by centrifugation for 5 min at 800 × g.
- Wash the resin twice with 10 bed volumes of urea buffer and
recover the resin by centrifugation, as in step 8. - Transfer the resin into a 15 mL tube. Elute the caspase with a
step gradient of imidazole in urea buffer (0–200 mM imidaz-
ole; 12 fractions; 1 bed volume per fraction) by suspending the
resin in buffer then by centrifugation for 5 min at 800 × g.
Full-Length Caspase-8
Expression Protocol
Full-Length Caspase-8
Purification Protocol
Dave Boucher et al.http://www.ebook3000.com