242

3. Radiolabeled L -[^35 S]methionine (PerkinElmer EasyTag™ Cat.

   NEG709A, or similar.

   
   


4. RNasin ® Ribonuclease Inhibitor (Promega).


5. Recombinant metacaspase protein stock solution. Prepare
   dilutions in an 8-well eppi strip at 4× the fi nal reaction concen-
   tration in buffer containing 50 % glycerol and 25 mM HEPES
   (pH 7.5). For recombinant Arabidopsis thaliana metacaspase
   4 (rAtMC4), we standardly use a dilution series (fi nal concen-
   tration) of 4.0, 1.0, 0.5, 0.25, 0.125, and 0.0 μM. Inactive
   rAtMC4, in which the active site cysteine is mutated to alanine
   (rAtMC4-C139A), acts as a negative control and is used at
   4.0 μM. Store at −20 °C.


6. 4× Reaction buffer: 200 mM HEPES pH 7.5, 600 mM NaCl,
   40 % glycerol, 200 mM CaCl 2. Add 40 mM DTT to an aliquot
   right before use.


7. 5× Laemmli buffer + EGTA: 250 mM Tris–HCl pH 6.8, 10 %
   (w/v) SDS, 20 % (v/v) glycerol, 0.1 % bromophenol blue,
   250 mM EGTA. Add 10 % β-mercaptoethanol to an aliquot
   right before use. This is aliquoted in an 8-well eppi strip.


8. 12 % Resolving gel: 12 % Acrylamide–Bis-acrylamide (29:1),
   375 mM Tris–HCl pH 8.8, 0.1 % (w/v) SDS.


9. 4 % Stacking gel: 4 % Acrylamide–Bis-acrylamide (29:1),
   125 mM Tris–HCl pH 6.8, 0.1 % (w/v) SDS.


10. SDS-PAGE Running buffer: 25 mM Tris base, 24 mM gly-
    cine, 0.1 % (w/v) SDS.


11. Whatman 3MM paper (Whatman).


12. SDS-PAGE gel dryer (Bio-Rad, Model 583 or similar).


13. 8-well eppi strips and multichannel pipette (volume 5–50 μl).


14. 96-well heat block or water bath at 30 and 85 °C.


15. Phosphor Imager™ 445 SI (Amersham Pharmacia Biotech
    Benelux) and Storage Phosphor Screen (Molecular Dynamics),
    or similar.

3 Methods

Recombinant metacaspases can be used for various assays, e.g.,

analysis of substrate specifi city, preparation of antibodies, crystallog-

raphy, mutagenesis for functional mapping. The most time- and

cost-effi cient method of production of recombinant metacaspase is

expression in bacterial cells. Since metacaspases undergo autopro-

cessing and subsequent self-inactivation in vitro, the conditions for

metacaspase expression and purifi cation must be adjusted to

decrease proteolytic activity. For instance, induction of expression

at low temperatures gives higher yield of purifi ed zymogen. While

3.1 Production
of Recombinant
Metacaspases

Elena A. Minina et al.

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