7- Washing buffer: 50 mM Tris at pH 8.0, and 0.5 M NaCl
(autoclave-sterilized). - 2× TY media (1 L): 16 g tryptone, 10 g Bacto yeast extract,
5 g NaCl (autoclave- sterilized), 50 μg/mL ampicillin or
10 μg/mL kanamycin, and 25 μg/mL chloramphenicol
(see Note 5 and Subheading 3.1.1.1 for antibiotic selection). - Z-VAD-fmk solution: 100 μM in DMSO (keep at −20 °C in
10 μL aliquots).
3 Methods
There is no caspase in E. coli. However, because some E. coli
proteins can be cleaved by caspases [ 13 ], it is not beneficial to the
bacteria to express caspases. Therefore, caspases are best expressed
using a system that leaks as little as possible, which is, in this case,
the pET system (EMD Millipore, formerly Novagen) in the
BL21(DE3) E. coli strain. This strain drives expression of the pro-
tein of interest via a T7 promoter. DE3 is a λ prophage carrying
the T7 RNA polymerase gene and the lacIq repressor. An IPTG-
inducible promoter drives the T7 RNA polymerase expression,
which is repressed by lacIq. Furthermore, supplemental repression
is obtained if the bacterium carries the pLysS plasmid, which
encodes the T7 lysozyme, a T7 RNA polymerase inhibitor. Upon
addition of IPTG to the growth medium, the lacIq repressor is
neutralized, and the T7 RNA polymerase is expressed. The poly-
merase concentration then overcomes the T7 lysozyme inhibition
and drives the T7 promoter that is found on the pET plasmid
encoding the caspase. Although not absolutely necessary, the use
of pLysS is recommended, as it will facilitate bacterial growth
before protein expression induction and can prevent the selection
of weakly expressing bacteria during the culture.
All full-length caspases must be expressed as C-terminally His-
tagged proteins. This requirement arises because most caspases
cleave themselves in the N-terminal domain, thus resulting in the
loss of the catalytic domain if the purification tag is at the
N-terminus. The N-termini of caspases contain regulatory domains
that can be masked by the addition of a nearby tag. Furthermore,
several groups have successfully fused fluorescent proteins at the
C-termini of caspases [ 14 – 18 ]. However, initiator caspases
expressed without the N-terminal domain can be purified as
N-terminal His-tag fusion proteins.
Aside from a few exceptions (e.g., full-length caspase-8), cas-
pases express as soluble proteins and can be purified from the sol-
uble fraction of a bacterial lysate without the use of detergents.
Along with the regular protocol for purifying soluble active
caspases (Subheading 3.1.1), protocols are provided for full-length3.1 Caspase
Expression and
Purification
Apoptotic Caspases Assays