Purification, characterization, and crystallization of trypanosoma metacaspases

McLuskey, K., Moss, C. X. and Mottram, J. C. (2014) Purification, characterization, and crystallization of trypanosoma metacaspases. Methods in Molecular Biology, 1133, pp. 203-221. (doi: 10.1007/978-1-4939-0357-3_13)

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: McLuskey, Dr Karen and Moss, Dr Catherine and Mottram, Professor Jeremy
• Authors: McLuskey, K., Moss, C. X., and Mottram, J. C.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Methods in Molecular Biology
• Publisher: Springer New York
• ISSN: 1064-3745