Caspases,Paracaspases, and Metacaspases Methods and Protocols

11

of crude inclusion bodies using centrifugation. Proteins are then

solubilized using guanidine, and denatured caspase-8 is purified

using two chromatography-based purification steps: (1) IMAC to

recover all His-tagged proteins, and (2) optional ion exchange chro-

matography to remove caspase-8 fragments and concentrate the

protein. Finally, refolding is performed by dialysis against an arginine

buffer. Although the mechanisms of arginine- assisted refolding are

not fully understood, it seems to reduce protein aggregation by

interacting with amino acid side chains, increasing the free energy of

protein–protein interactions, and increasing the stability and solubil-

ity of denatured proteins [ 19 – 22 ].

The procedure described below has been used to generate

minute amounts of full-length caspase-8 fused at the C-terminus

with yellow fluorescent protein (YFP) (Fig. 3 ). The recovery of

YFP fluorescence was used as a mean to assess various refolding

protocols, and the presence of YFP does not alter the enzymatic

properties of the caspase. As shown in Fig. 3a, IMAC produces a

116

10 20 4050

0 50

7090100

100150200250300350400 mM NaCl

120140 160180200 200 mM imidazole

97.466.2

45

31

21.5

14.4

6.5

kDa

200

97.4^116

66.2

45

31

21.5

14.4

6.5

kDa

0

1

2

3

4

5

6

7

8

0 100 200 300 400 500

Afc released (nM/s)

AcIETD-Afc (mM)

0

400

800

1200

1600

450 550 650 750

Relative fluorescence

Coomassie stain Wavelength (nm)

Coomassie stain

a 527 nm

b

c

d

Fig. 3 Full-length caspase-8 production. (a) IMAC purification (Subheading 3.1.2) of denatured full-length
caspase-8 C-terminally fused to YFP (~84 kDa; arrowhead ). Proteins were eluted using a step gradient of
imidazole (indicated above each lane). The procedure results is a protein preparation that is >80 % pure.
(b) DEAE anion exchange chromatography of denatured full-length caspase-8 C-terminally fused to YFP
(arrowhead). This results in a protein preparation that is >90 % pure. (c) Following refolding, the typical fluo-
rescence spectrum of YFP is recovered showing a maximum emission of 527 nm. (d) The activity of caspase-8
is also recovered as demonstrated by the typical Michaelis–Menten substrate saturation curve. These data are
consistent with a KM and kcat of 4.4 μM and 0.4 s−1, respectively

Apoptotic Caspases Assays