Identification and analysis of Cryptosporidium Glutathione Transferase.
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Date
2021
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Abstract
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.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.