Toxoplasma gondii: p450 bacterial expression and protein purification

Human infection with the protozoan parasite Toxoplasma gondii can cause serious disease in immunocompromised individuals and the developing fetus. Human infection is characterized by the tachyzoite (acute infection) and bradyzoite (encysted form, latent infection) stages. Current therapies are ineffective against the bradyzoite stage of infection because this form replicates very slowly. Cytochrome P450 enzymes perform an array of monooxygenation reactions in diverse pathways in many organisms. Recent literature indicates that P450 inhibitors decrease the number of bradyzoites in T. gondii infected mice. We identified the gene for a novel P450 protein in the Toxoplasma genome. In-frame fusion of the Toxoplasma P450 to yellow fluorescent protein (YFP) shows colocalization of the P450 with a mitochondrial marker. The goal of my study is to express large quantities of the T. gondii P450 protein in Escherichia coli. Using SDS PAGE gels, I have established the optimal conditions for expressing the P450: growth at 30°C in BL21-CodonPlus E. coli with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction for 3 hours. The protein has a carboxy-terminal His₆ tag that allows protein purification using a nickel column. I am currently working on purification of the protein. When assayed by absorbance spectroscopy, my purified protein should show a Soret peak at 450 nm, a characteristic of P450 cytochromes. The protein will be used for crystallography. Resolving the structure of the Toxoplasma P450 will be important to identify new P450 inhibitors.