37In E. coli, a fraction of the protein subunits will reassemble into
a fully cleaved dimer. This approach can be particularly useful to
obtain fully processed but inactive or poorly active caspase
mutants.
More elaborate expression schemes have been used to generate
caspase dimers with different catalytic domains [ 35 ]. These het-
erodimeric caspases were produced by co-expressing two different
caspase-7 molecules, each carrying a different C-terminal purifica-
tion tag, for example, a His-tag on one caspase-7 molecule and a
FLAG-tag on the other. Briefly, after IMAC, the purified caspase
was purified on anti-FLAG-coupled IgG beads and eluted with
free FLAG peptide. The resulting product is a true hybrid dimer of
caspase-7. This method allows for the detailed characterization of
an executioner caspase [ 35 ].
Recently, an engineered caspase-7 that is sensitive to redox
conditions has been described [ 56 ]. In oxidizing conditions, two
substrate-binding loops remain linked by a disulfide bound between
two designed cysteine residues, inactivating the caspase. In reduc-
ing condition, the disulfide bond is broken, and the caspase
becomes fully active.
Another recent study reports the presence of additional deter-
minants on caspases involved in substrate cleavage [ 32 ]. Those
determinants, called exosites, are located outside the substrate-
binding pocket and add an additional layer of specificity to the
enzyme. Currently, only one exosite has been characterized, but
much evidence in the literature suggests the presence of other
exosites. Allosteric sites also exist in caspases [ 57 – 59 ]. The identi-
fication of exosites and allosteric sites brings opportunities to
develop more specific caspase inhibitors.
In conclusion, the expression and purification of caspases have
been useful since the time these enzymes were discovered more
than 20 years ago and will remain a valuable and necessary tool to
study the many processes in which caspases are involved.References
- Cerretti DP et al (1992) Molecular cloning of
the interleukin-1β converting enzyme. Science
256:97–100 - Thornberry NA et al (1992) A novel heterodi-
meric cysteine protease is required for
interleukin- 1beta processing in monocytes.
Nature 356:768–774 - Yi CH, Yuan J (2009) The Jekyll and Hyde
functions of caspases. Dev Cell 16:21–34 - Thornberry NA et al (1997) A combinatorial
approach defines specificities of members of
the caspase family and granzyme B.
Functional relationships established for key
mediators of apoptosis. J Biol Chem 272:
17907–17911
5. Stennicke HR, Renatus M, Meldal M, Salvesen
GS (2000) Internally quenched fluorescent
peptide substrates disclose the subsite preferences
of human caspases 1, 3, 6, 7 and 8. Biochem J
350:563–568
6. Lavrik IN, Golks A, Krammer PH (2005)
Caspases: pharmacological manipulation of cell
death. J Clin Invest 115:2665–2672
7. Garcia-Calvo M et al (1998) Inhibition of human
caspases by peptide-based and macromolecular
inhibitors. J Biol Chem 273:32608–32613
8. Fuentes-Prior P, Salvesen GS (2004) The pro-
tein structures that shape caspase activity, speci-
ficity, activation and inhibition. Biochem J 384:
201–232
Apoptotic Caspases Assays