Abstract
Metacaspases are cysteine peptidases found in trypanosomes but absent in mammals, and despite being distantly related to the mammalian caspases they show significant disparity in their cellular and enzymatic functions. The genome of the parasitic protozoa Trypanosoma brucei (the causative agent of African sleeping sickness) encodes five 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 divergence from the caspases, paracaspases, and other Clan CD cysteine peptidases (or vice versa), we purified 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 TbMCA2C213A. 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 TbMCA2C213A.
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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.
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McLuskey, K., Moss, C.X., Mottram, J.C. (2014). Purification, Characterization, and Crystallization of Trypanosoma Metacaspases. In: V. Bozhkov, P., Salvesen, G. (eds) Caspases,Paracaspases, and Metacaspases. Methods in Molecular Biology, vol 1133. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0357-3_13
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DOI: https://doi.org/10.1007/978-1-4939-0357-3_13
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