Caspases,Paracaspases, and Metacaspases Methods and Protocols

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The central cell-killing pathway in C. elegans (EGL-1 –| CED-9

–| CED-4 → CED-3) is well characterized [ 4 ]. CED-3 acts at the

terminal step of this pathway and, as a protease, is expected to

cleave numerous protein substrates to promote cell death execu-

tion [ 11 , 12 ]. To identify CED-3 protease substrates, several dif-

ferent approaches have been employed [ 13 – 15 ]. Candidate CED-3

substrates identifi ed from biochemical screens or genetic screens

usually need to be confi rmed as true in vitro substrates before

time-consuming functional studies are performed. Therefore, a

robust in vitro CED-3 caspase cleavage assay is critical for deter-

mining candidate CED-3 substrates [ 16 – 20 ]. To perform this

experiment, successful purifi cation of the CED-3 protease is

essential.

Successful purifi cation of active recombinant CED-3 can be

achieved through bacterial expression [ 18 ]. In C. elegans , CED-3

is initially expressed as an inactive proenzyme of molecular weight

56 kDa. In dying cells, the CED-3 proenzyme is induced to

undergo self-cleavage in a CED-4-dependent manner, leading to

the generation of 24, 17, and 13 kDa cleavage products, and

becomes self-activated [ 18 , 21 ]. While many mammalian caspases

are commercially available, CED-3 is not. In general, purifi cation

of a protease without losing its activity is challenging compared

with preparation of other recombinant proteins. This has been a

major issue in purifi cation of CED-3, because CED-3 tends to lose

its proteolytic activity during the purifi cation process.

Here, we describe the method for purifi cation of active CED-3

and further analytical assays using the purifi ed CED-3 protease.

2 Materials

1. A pET-21 vector (Novagen) with a six-histidine affi nity tag
   (His 6 tag) for C-terminal fusion can be used for expression of
   CED-3 ( see Notes 1 – 3 ). Full-length CED-3 cDNA without a
   stop codon can be amplifi ed by the polymerase chain reaction
   (PCR) and inserted into the vector via appropriate multiple
   cloning sites just before the His 6 coding region.


2. Bacterial strain BL21 (DE3).


3. 2 L of LB medium with an appropriate antibiotic in a 4 L fl ask.


4. 2 ml of IPTG stock (1 M).


5. 37 °C shaking incubator and 25 °C shaking incubator.


6. 4 °C high-speed centrifuge.


7. 250 ml or 500 ml centrifuge bottles for collecting bacterial
   cells.


8. Sonicator with a fl at tip ( see Note 4 ).

2.1 Purifi cation of
Recombinant CED-3

Eui Seung Lee and Ding Xue

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