203Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_13, © Springer Science+Business Media New York 2014
Chapter 13
Purifi cation, Characterization, and Crystallization
of Trypanosoma Metacaspases
Karen McLuskey , Catherine X. Moss , and Jeremy C. Mottram
Abstract
Metacaspases are cysteine peptidases found in trypanosomes but absent in mammals, and despite being
distantly related to the mammalian caspases they show signifi cant disparity in their cellular and enzymatic
functions. The genome of the parasitic protozoa Trypanosoma brucei (the causative agent of African sleep-
ing sickness) encodes fi ve metacaspases: TbMCA1-TbMCA5. Of these TbMCA2, TbMCA3, and TbMCA5
are active cysteine peptidases expressed in the bloodstream form of the parasite. To investigate the
structure–function relationship of the trypanosome metacaspases and the structural basis for their diver-
gence from the caspases, paracaspases, and other Clan CD cysteine peptidases (or vice versa), we purifi ed
and characterized TbMCA2 and determined the three-dimensional structure of an inactive mutant using
X-ray crystallography. The methods presented in this chapter describe the recombinant expression of active
TbMCA2 and inactive TbMCA2 C213A. The protocols produce large amounts of recombinant protein for
use in structural, biochemical, and kinetic studies and include detailed information on how to produce
diffraction quality crystals of TbMCA2 C213A.
Key words Trypanosome , Metacaspase , TbMCA2 , Parasite , X-ray crystallography , Protein purifi ca-
tion , Enzyme assay , Cysteine protease1 Introduction
Metacaspases are cysteine peptidases, which are distantly related to
the mammalian caspases. They share a conserved histidine/cysteine
(His/Cys) catalytic dyad and a basic caspase hemoglobinase fold,
but metacaspases are only found in phylogenetic kingdoms lacking
caspases such as plants, yeast, and lower eukaryotes including try-
panosomes. A number of plant metacaspases have been shown to
play a role in cell death pathways [ 1 ], suggesting a link with the cell
death control exhibited by the caspases, which are key players in
mammalian programmed cell death. However, trypanosomatid par-
asitic protozoa such as Trypanosoma brucei and Leishmania , the
causative agents of Human African Trypanosomiasis and Leish-
maniasis, respectively, appear to lack such regulated cell death [ 2 ].