Trypanosoma cruzi, the cause of Chagas disease, affects nearly 18 million people in South and Central America, of whom 50,000 will die each year. One possible target for new drugs to treat Chagas disease is the sterol biosynthesis pathway. T. cruzi uses the sterol biosynthesis pathway to generate ergosterol, a molecule used to stabilize the cell membrane. Sterol biosynthesis enzymes have been successfully exploited as drug targets for other diseases, such as leishmaniasis and fungal infections. One enzyme which has been the focus of drug development for Chagas disease is sterol 14-demethylase, a cytochrome P450 enzyme. This molecule is oxidized and inactivated during sterol biosynthesis and thus requires the presence of a P450 reductase (P450R) to return it to its active state. The goal of this project was to clone, express, and purify the functional T. cruzi P450 reductase so that it could be used in concert with the T. cruzi sterol 14-demethylase enzyme in assays to screen for novel inhibitors of the pathway. Identification of compounds that inhibit T. cruzi sterol 14-demethylase will potentially lead to new drugs for the treatment of Chagas disease. The P450R gene from Trypanosoma brucei, a related trypanosome, had previously been cloned by the lab and served as the starting point for my studies, as we predicted it would have cross activity with the T. cruzi sterol 14-demethylase. This gene was ligated into a pET 100 TOPO vector regulated with a lac promoter and containing a six histidine-tag for purification. We attempted to express the protein in BL21* E. coli. A cytochrome C assay was used to identify activity of the protein. Although some activity was identified, purification using a nickel bead column was unsuccessful. The T. brucei P450R gene was recloned from this plasmid and inserted into an alternative E. coli expression vector, pET 14b. Candidate T. cruzi P450R genes TcR60, TcR70, and TcR120 were cloned from genomic T. cruzi DNA and inserted into the pET 14b vector by ligation independent cloning (LIC). We attempted to express these four genes in BL21* E. coli. An appropriate 68 kDa protein representing TcR120 was expressed, and protein was confirmed to have the expected 6-histidine tag on a Western blot using nickel-conjugated alkaline phosphatase. This protein, however, did not appear to have active reductase capabilities as determined by a cytochrome C assay. Ongoing expression studies are underway with recombinant expression of the additional T. cruzi P450R candidate genes to establish if one of these can yield functional reductase activity.
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