Identification and analysis of Cryptosporidium Glutathione Transferase.
Mfeka, Sizamile Mbalenhle.
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Cryptosporidiosis, caused by Cryptosporidium spp. is a gastrointestinal disease which gives rise to severe life threatening complications in immunecompromised patients. The disease causing parasite has a proficient defense system against xenobiotic compounds and substances that renders the only drug designed to treat the gastroenteritis infection inefficient in immune compromised patients. This defense system includes a phase II enzyme called Glutathione Transferase (GST) which detoxifies a wide range of oxidant based substrates. The overexpression of this protein in multi drug resistant cases and its presence in multiple stages of the parasites life cycle highlights the parasites dependence and utilization of the GST protein thus making it a suitable therapeutic target. This study was then set out to determine characteristic features of Cryptosporidium GSTs in comparison to well studied GSTs using molecular biology and bioinformatics tools. A genome wide search was performed across multiple protein databases to mine the Cryptosporidium GST. The 15 Cryptosporidium spp. found to possess full length proteins were compared amongst themselves within the species and against other species using phylogenetic analyses. This led to the discovery of three novel classes of Cryptosporidium GST based on amino acid sequence identity. The classes were named Gamma, Psi and Vega GSTs. The GSTs varied in amino acid length, and secondary structure characteristics determined through homology modeling. In comparison to preexisting GSTs, the Psi and Vega class GSTs did not have the typical active site Tyr7 found in most cytosolic GST, furthermore the Vega class GST also did not have the typical thioredoxin like fold conserved in the N-terminal region of all GSTs. The Gamma class GSTs were found to most resemble pre existing GSTs consisting of the typical thioredoxin fold and the active site Tyr7 and thus selected for expression and purification studies. pET, pCOLD1 and pCOLDTF vectors were used to determine a suitable vector to facilitate the expression of a soluble gamma class GST in Escherichia coli. pCOLDTF which utilizes cold shock proteins at low temperatures and a chaperone called trigger factor assisted in the recombinant expression of the gamma class GST resulting in a protein with the monomer size of ~50 kDa, which is double that of existing GSTs. This is owed to by the N-terminal and C-terminal extensions that the protein possesses. The protein was purified to homogeneity using affinity chromatography and size exclusion chromatography. The resulting protein was found to be dimeric under native conditions.