25substrate-binding pocket, active site titration requires conditions
that force inhibition by Z-VAD-fmk. Caspase-2 can be successfully
titrated using 2 μM of estimated concentration of the peptidase
and serial dilution of Z-VAD-fmk started at 20 μM.
Caspase-3
Caspase-3 is the easiest executioner caspase to produce and purify.
This enzyme is highly active and titrates well using the Z-VAD-fmk
inhibitor. Caspase-3 is the caspase with the highest intrinsic activity
and displays the highest activity on AcDEVD-Afc and a wide vari-
ety of other sequences [ 4 , 5 , 23 ]. Consequently, one must be care-
ful when studying caspase activity in cells and in vitro because
caspase-3 can often overpower other caspases, even on their best
substrates. To express the zymogen form of caspase-3, a short time
of expression (25–30 min) is used to prevent auto- proteolysis, and
purification of the protein must be performed immediately follow-
ing expression (without freezing), using a minimal amount of
Chelating Sepharose resin (0.5–1.0 mL). Because yields are low, it
is recommended to use at least 4 L of culture.
Caspase-6
Better yields are obtained if caspase-6 is expressed slowly with low
IPTG concentration (0.02–0.05 mM) and long expression time
(16–20 h). The relatively long expression time also ensures removal
of the N-terminal peptide and efficient cleavage of the linker.
Caspase-6’s activity is not stable over time. Consequently, cas-
pase-6 must be used quickly after thawing. For example, for titra-
tion, the inhibition step is performed for 15 min instead of 30 min
to enable the measurement of a proper level of remaining activity.
Caspase-7
Eight hours of expression time for caspase-7 is enough to obtain a
fully processed enzyme (N-terminal peptide removed and linker
cleaved). Longer expression time results in unwanted cleavage at
Asp192 (Asp291 according to caspase-1 nomenclature) that inac-
tivates the enzyme [ 35 ]. It is also important to pay particular atten-
tion to the pH of the buffers that are used for the purification of
some specific forms of caspase-7. Indeed, the zymogen form has a
pI of 5.7 and can be efficiently purified with buffers at pH 8.0.
However, for the mature form or for any forms that do not carry
the N-terminal peptide (residues 1–23), the pI is 8.3. Thus, it is
suggested to use purification buffers at pH 7.2 to limit protein
precipitation. See caspase-3 remarks for the expression of the
zymogen form of caspase-7.
Caspase-8
The truncated form of caspase-8 expresses well as a soluble enzyme
in E. coli and is easily purified using the procedure described in
Subheading 3.1.1. The absence of its DEDs does not affect cas-
pase- 8 substrate specificity on small peptidic substrates [ 36 ].
However, because full-length caspase-8 aggregates, this form is
Apoptotic Caspases Assays