Caspases,Paracaspases, and Metacaspases Methods and Protocols

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substrate, site-directed mutagenesis of the predicted CED-3 cleavage

sequence is usually performed. Asp to Glu substitution or Asp to

Ala substitution at the P1 position blocks CED-3 cleavage [ 16 –

20 ]. The mutated DNA template is used for the in vitro translation

and cleavage assays, and the CED-3 cleavage pattern of the mutant

protein is compared with that of the wild-type protein. If CED-3

cleavage of the mutant protein is blocked or greatly reduced, then

the mutated site is likely a CED-3 cleavage site. However, if the

substrate protein can be purifi ed and is soluble, we would recom-

mend to perform microsequencing analysis of the amino terminus

of the cleavage product following in vitro cleavage with purifi ed

CED-3 to determine the caspase cleavage site.

1. Prepare at least 10 μg of the purifi ed substrate protein from
   E. coli.


2. Incubate 10 μg of the purifi ed protein with 1 μg of purifi ed
   CED-3 in 50 μl CED-3 reaction buffer.


3. Incubate the reaction at 30 °C for 4–10 h.


4. For the negative control, use the elution buffer that was used
   for CED-3 purifi cation.


5. Stop the reaction by adding SDS loading buffer and then heat
   the samples at 95 °C for 5 min.


6. Resolve the protein samples on SDS-PAGE. Load purifi ed
   CED-3 alone to distinguish the CED-3 cleavage products
   from the 13 and 17 kDa CED-3 subunits.


7. Transfer the proteins on the SDS-PAGE gel to a PVDF mem-
   brane using a wet or semidry transfer apparatus.

Fig. 1 In vitro cleavage assay. A pET-3a-CED-9 vector was used to synthesize

[^35 S] Methionine-labeled CED-9 using the TNT rabbit reticulocyte lysate system.

The molecular weight of full-length CED-9 is 31 kDa. When incubated with puri-

fi ed CED-3, full-length CED-9 disappeared, and instead, 7 and 24 kDa fragments

were observed

Eui Seung Lee and Ding Xue

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