220
- All fractions should be checked by SDS-PAGE to identify the
protein, as the elution profi le may change depending on the
temperature and pH of the buffers. This protocol is good for
inactive TbMCA2 C213A , but other forms of this enzyme and
other T. brucei metacaspases may require additional purifi ca-
tion steps. - SimplyBlue SafeStain (Invitrogen) can be disposed of safely by
fl ushing down the sink and the gels destained using ddH 2 O.
Other types of Coomassie stain may require specifi c disposal
methods and destaining protocols, and if chosen these should
be investigated. - The extinction coeffi cient can be calculated from the primary
sequence using tools such as ProtParam ([ 21 ], http://web.
expasy.org/protparam/ ). The extinction coeffi cient of
TbMCA2 C213A , including the linker and His-tag is
29,910 M −1 cm −1 and the Beer–Lambert Law ( A = Ecl ) can
then be used to calculate the relative protein concentration
where A = A 280 , E = extinction coeffi cient, c = concentration,
and l = path length of the cell. While this method may not give
an exact measure of protein concentration (relying on the
absorbance from the Tyr and Trp residues) it is suffi ciently
accurate and reproducible for preparation of samples for crys-
tallization trials. - Purifi ed T. brucei metacaspases should be stored at 4 °C rather
than at −20 °C, as freeze–thaw cycles are known to reduce pep-
tidase activity and affect the crystallizability of TbMCA2 C213A.
In addition, TbMCA2 C213A should be used in crystallization
trials as soon as possible after purifi cation as any delay in this
will affect the success rate of reproducing crystals. - In the case of TbMCA2 C213A , the crystallization solution can
be used as an appropriate stabilizing solution for seeding. - One enzyme unit ( U ) is the amount of enzyme that catalyzes
the reaction of 1 μmol of substrate. - The incubation time is enzyme specifi c and will have to be
optimized for different proteins and possibly different batches
of the same protein. The given times are based on assays using
active recombinant TbMCA2.
Acknowledgements
This work was supported by Wellcome Trust Grant 091790 and
Medical Research Council Grant 0700127. The Wellcome Trust
Centre for Molecular Parasitology is supported by core funding
from Wellcome Trust Grant 085349.Karen McLuskey et al.http://www.ebook3000.com