The expression vector pET28a+ (Novagen) was used for the
protein expression of metacaspases from T. brucei. However,
other expression vectors may be used provided potential cleav-
age sites in the linker are considered ( see Note 1 ).
Most of the T. brucei metacaspases express well in both BL21
(DE3) and Rosetta (DE3) cell strains although TbMCA4 gen-
erally produces a much higher yield in Rosetta (DE3).
Varying cell lines and vectors will require different antibiotics
and in general a 1,000× stock solution is prepared in ddH 2 O
and stored at −20 °C, until required.
T. brucei metacaspases generally express very well giving a
purifi ed yield (after size exclusion chromatography) of around
5 mg from a 250 mL cell culture and these amounts can be
scaled up/down as required.
Centrifugal concentrators can be used to exchange the buffer
for any protein that can tolerate the procedure and T. brucei
metacaspases are generally amenable to it. The protein sample
should be concentrated to a minimum volume and diluted
back to the starting volume in the new buffer, this should be
repeated two/three times to ensure buffer exchange.
It might be useful to vary the PEG concentration in the crys-
tallization experiments but in general seeding was the most
effective method for crystal improvement.
These may be useful for other T. brucei metacaspases, but for
the inactive TbMCA2 C213A mutant the stated crystallization
condition should be most successful.
The excitation and emission maxima for the fl uorescent
molecule (AMC) are 345 nm and 445 nm, but wavelengths at
either side of these values can be used, albeit with decreased
fl uorescence ( see http://www.invitrogen.com/site/us/en/
home/Products-and-Services/Applications/Cell-Analysis/
Labeling-Chemistry/Fluorescence-SpectraViewer.html ).
Expression plasmids for the proteins described in this chapter,
can be identifi ed by their plasmid numbers (pGL): Active
TbMCA2, pGL1573; TbMCA2 C213A , pGL2128; TbMCA3,
pGL1593; TbMCA4, pGL1967.
LB (Luria–Bertani) medium can also be used for recovery and
a 30 min incubation may also suffi ce.
It is advisable to plate out two different volumes on to the
LB-Agar plate and keep excess cells overnight at 4 °C in order
to plate out an optimized volume the next day, if required.
Using auto-induction media results in a large amount of pro-
teins in the cell lysate. In order to recover a reasonable amount
of T. brucei metacaspases from the IMAC column it is better to
limit the amount of cell culture used to a maximum of 500 mL.
