Cloning of the Gene, Purification as Recombinant Protein and Functional Characterization of Leishmania mexicana Cytochrome b5 Reductase

• Main Author: Azhari, Ala
• Format: Others
• Published: Scholar Commons 2012
• Subjects: detoxification of xenobiotics; drug resistance; Leishmaniasis; NADH; protein expression; Molecular Biology; Parasitology; Public Health
• Online Access: http://scholarcommons.usf.edu/etd/4282; http://scholarcommons.usf.edu/cgi/viewcontent.cgi?article=5478&context=etd

Leishmania are protozoan parasites that are transmitted by a sand fly vector. These parasites affect not only humans but also wild animals including domestic dogs and rodents, which form an additional challenge and public health problem to control the disease. Leishmaniasis is an important disease with worldwide distribution, including Saudi Arabia, the Middle East, and other tropical and subtropical areas around the world. Due to the expansion of irrigation and agricultural activities, more exposure to sand fly occurs, which leads to the expansion of leishmaniasis infections as newly emerging disease. Emerging drug resistance in leishmaniasis is an additional problem, contributed by enzymes involved in the detoxification of pharmacological agents and other xenobiotics. Cytochrome b5 reductase (Cb5r) has a high pharmacological significance because of its essential role in fatty acid elongation, biosynthesis of cholesterol (humans) or ergosterol (Leishmania, fungi), and cytochrome P450-mediated detoxification of xenobiotics. Leishmania Cb5r has seven different isoforms whereas human has only one. Cb5r-7 isoform in Leishmania has closest homology to the human Cb5r. The three specific aims of this thesis project are focusing on (i) cloning of the Cb5r-7 isoform from Leishmania mexicana, (ii) its purification as recombinant His-tagged protein from E.coli, and (iii) its functional characterization as potential pharmacological target against Leishmania.