Caspases,Paracaspases, and Metacaspases Methods and Protocols

243

prolongation of induction time leads to accumulation of high amount

of metacaspase in bacterial cells, this also induces intermolecular

cleavage of the protease signifi cantly reducing the yield of enzymati-

cally active protease.

Both N- and C-terminal tags can be used for purifi cation of

metacaspases without disturbing their proteolytic activity.

Introducing metacaspase-specifi c cleavage sites should be avoided

while cloning.

We have established a protocol for producing recombinant

Norway spruce metacaspase mcII-Pa using His-tagged system.

This system allows purifi cation under either native or denaturing

conditions. Although denaturing conditions give higher amounts

of pure zymogen that can be used for raising antibodies, they are

not recommended for producing metacaspases for enzymatic activ-

ity assays due to the need for protein refolding.

1. To avoid expression of metacaspase before induction, clone
   metacaspase gene under control of lac promoter and make
   sure that either the plasmid with the metacaspase gene or the
   E. coli strain contain lacI gene. For example, we commonly use
   pET11 vectors (Novagen) or pDEST17 vector (Gateway,
   Invitrogen ® ) with BL21(DE3) Rosetta (Novogen) or
   BL21(DE3)pLysE (Invitrogen) cells. It is preferable to use
   freshly transformed cells to avoid any random mutations in the
   expressed gene.


2. Grow bacterial culture overnight in a small volume at 37 °C.


3. Next morning dilute the culture till OD 600 = 0.1 and incubate
   it at the same growth conditions until OD 600 reaches 0.4–0.6.


4. Let the cell culture cool down on ice for 10–15 min.


5. Add IPTG to the fi nal concentration 0.5 mM and incubate the
   culture on a shaker in the darkness at 28 °C for 2 h or over-
   night at 16 °C.


6. Pellet down the cells for 15 min at 4,000 × g at 4 °C. The cell
   pellet can be stored at −20 °C for at least several weeks with-
   out affecting the proteolytic activity of metacaspase.


7. Gently resuspend the cells in a small volume of cold sonication
   buffer and lysate by sonicating at the amplitude 1 μm for 20
   pulses: 20 s on/20 s off. Make sure that the cell suspension is
   not warmed up during sonication. Pellet the cell debris by cen-
   trifugation for 10 min at 17,000 × g at 4 °C. Add imidazole,
   pH 8.0 to the cleared lysate to the fi nal concentration 10 mM
   and β-mercaptoethanol to the fi nal concentration 10 mM.


8. Alternatively, the cells can be resuspended in binding buffer
   and lysed using French press.


9. Load the cell lysate on Ni-NTA column and let it pass through
   the column several times. Incubating for 2 h at 4 °C with
   Ni-NTA resin can additionally increase the yield.

3.2 Purifi cation
Under Native
Conditions

Plant Metacaspases