Caspases,Paracaspases, and Metacaspases Methods and Protocols

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caspase-8 fused at the C-terminus to YFP. These protocols describe
expression and purification from inclusion bodies, followed by
refolding the protein to recover its activity (Subheading 3.1.2).

The protocols are described for 1–2 L of bacterial culture, which is
generally sufficient to produce enough caspase for most biochemi-
cal characterization. The protocols can be scaled up to accommo-
date larger expression volumes and protein yields. However, the
centrifugation required to harvest bacteria may limit the total man-
ageable volume when the zymogen forms of caspase-3 or caspase-7
are produced because of short expression time (<30 min). If this is
the case, several smaller expression cultures should be planned
unless there is access to a high-capacity rotor.
The procedure described below generates significant amounts
of many caspases (Fig. 2 ). As shown in Fig. 2a, the absorbance pro-
file of the imidazole-eluted fraction at 280 nm shows a typical
broad peak indicative of His-tagged protein, in this case, the cata-
lytic mutant of caspase-7 (35 kDa). If the yield is high, as is usually

3.1.1 General Protocol
for Caspase Production
in E. coli


Coomasie stain

kDa
200
116/97.4
66.2
45
31
21.5
14.4
6.5

200
116/97.4
66.2
45
31
21.5
14.4
6.5

Fraction #

kDa (^628101214161820223678910)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
a
bc
12345678910111213141516171819202122232425
Absorbance @ 280 nm
Fraction #
Caspases
Coomasie stain
Fig. 2 Caspase purification. Caspases were expressed and purified according to the protocol described in
Subheading 3.1.1. (a) Absorbance profile at 280 nm of each fraction for the expression of C-terminal His-
tagged caspase-7 C285A mutant (catalytic cysteine mutated to alanine; Subheading 3.5 for details). The white
trace represents the absorbance of the buffer alone. (b) Typical SDS-PAGE analysis of imidazole-eluted
caspase- 7 C285A (different purification than in (a). (c) SDS-PAGE analysis of various caspase preparations.
Caspase-2 and caspase-9 lack the CARD; caspase-8 and caspase-10 lack DEDs
Dave Boucher et al.
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