36
are too diluted for analysis, set up larger sample volumes and
precipitate proteins with 10 % trichloroacetic acid (TCA) for
2 h at −20 °C. The proteins are then recovered by centrifuga-
tion for 30 min at 18,000 × g at 4 °C and washed twice with
acetone before solubilizing the proteins in 1× SDS- PAGE
loading buffer.5 Discussion
The protocols described in this chapter permit the expression,
purification, and basic characterization of apoptotic caspases. The
techniques and instrumentation that are necessary to perform
these procedures are available to most laboratories. Therefore, it is
relatively easy to biochemically assess most caspase mutants to
ensure that enzymatic properties are not significantly altered prior
to performing cellular studies.
Caspases are versatile enough to allow the production of vari-
ous molecular forms. Indeed, by using cleavage site mutants, vary-
ing expression conditions, and using biochemical tricks (e.g.,
kosmotropic salts), all known molecular forms of caspases that
occur in cells can be produced. It has been suggested recently that
cleavage of the initiator caspase-8 regulates proteolysis of key sub-
strates, but not during necroptosis [ 51 ]. Thus, the ability to pro-
duce active but uncleaved full-length caspase-8 will be a valuable
tool to characterize these two molecular forms of initiator caspase.
Further protein engineering has allowed the “on-demand” activa-
tion of initiator caspases in cells using chemically induced dimer-
ization (CID) domains [ 36 , 51 – 53 ].
The linker that separates the large and small caspase subunits is
very flexible and is thus amenable to modification. Methods to
activate executioner caspases that bypass initiator caspases have
been developed in which an exogenous peptidase is used to cleave
the caspase. The tobacco etch virus (TEV) protease is a good can-
didate for this type of experiment, as this peptidase is highly spe-
cific and has no substrates in human cells. The approach uses
exogenously expressed TEV protease to cleave an engineered
cleavage site in the linker of the catalytic domain [ 52 , 54 ]. However,
experimenters must be careful with the location used to introduce
the cleavage site because the linker has specific determinants that
are involved in stabilization of the active caspase form [ 38 , 55 ].
This approach has also been used to study the specificity of execu-
tioner caspases in cells [ 54 ]. Other peptidases, such as thrombin,
have been proposed as exogenous peptidases to cleave caspases
[ 41 ], but their cellular utilization must be carefully assessed.
If the remnants of an engineered cleavage site are poten-
tially detrimental, the large and the small caspase subunits can
be expressed separately on the same vector or by co-expression.Dave Boucher et al.http://www.ebook3000.com