Caspases,Paracaspases, and Metacaspases Methods and Protocols

105

5. As a negative control, the same concentration of DMSO as in
   the reaction solution can be used, as the substrate has been
   dissolved in DMSO. 5 μM AMC can be used as a positive
   control.

A candidate CED-3 substrate can be synthesized in vitro using the

translation and transcription (TNT)-coupled reticulocyte lysate kit

(Promega). Full-length cDNA encoding the candidate substrate

can be cloned into a vector carrying the bacterial T7, T3, or SP6

promoter, such as pGEM-4Z, pET, or pBluescript vectors. To

visualize the CED-3 cleavage products, the substrate protein

should be labeled with [^35 S] Methionine. The reaction mixture and

procedure are as follows:

1. Prepare in vitro translation reaction mixture (10 μl reaction)
   ( see Note 11 ):
   (a) 5 μl of TNT reticulocyte lysate.
   (b) 0.4 μl of TNT reaction buffer.
   (c) 0.2 μl of polymerase (T7, T3, or SP6 polymerase depending
   on the promoter).
   (d) 0.2 μl of amino acids mixture without Methionine.
   (e) 0.4 μl of [^35 S] Methionine (1,000 Ci/mmol at 10 mCi/ml).
   (f) 0.2 μl of RNasin (ribonuclease inhibitor, 40 unit/μl).
   (g) 200 ng of the plasmid DNA template.
   (h) Add RNase-free water to a fi nal volume of 10 μl.


2. Incubate the reaction at 30 °C for 90 min.


3. Add 5 μl of 2× CED-3 reaction buffer to 1–4 μl of the labeled
   product.


4. Add 1 μl of the purifi ed CED-3.


5. Add distilled water to bring the fi nal volume to 10 μl.


6. Incubate at 30 °C for 1–4 h.


7. Stop the reaction by adding SDS loading buffer and heating at
   85 °C for 10 min.


8. Prepare a positive control: CED-9 is known to be one of the
   best CED-3 substrates and can be used as a positive control
   for the in vitro CED-3 cleavage assay [ 17 ]. If purifi ed CED-3
   has good activity, full-length CED-9 (31 kDa) should be com-
   pletely digested to generate 7 and 24 kDa cleavage products
   (Fig. 1 ).

CED-3 is known to recognize the amino acid sequence, Asp or

Glu-X-X-Asp (also called P4-P3-P2-P1; X can be any amino acid)

[ 18 ], and cleave right after the P1 Asp residue, which is absolutely

conserved. Therefore, to identify the cleavage site(s) of a CED-3

3.3 In Vitro Cleavage
Assay Using
Substrates Labeled by
[^35 S] Methionine

3.4 Determination of
the CED-3 Cleavage
Site in the CED-3
Substrate

In Vitro CED-3 Assays