Expression and Purification of the Enzyme Pyruvate Phosphate Dikinase from Leishmania major as a Chemotherapeutic Target

Leishmania major is the ethological agent of cutaneous leishmaniasis (CL) that affects millions of people worldwide. Currently, there are limited medications to treat parasites especially since new drug resistant strains have been discovered. Therefore, the developments of new drug therapies are necessary. The pyruvate phosphate dikinase (PPDK) works in the parasite's energetic metabolism and is not found in mammalian cells, therefore PPDK could be selectively inhibited. Based on this, we hypothesize that LmPPDK could be an excellent chemotherapeutic target and its inhibition would be lethal for the parasite. In this study we propose to express and purify LmPPDK in order to develop a high throughput screening assay. The LmPPDK gene was amplified by polymerase chain reaction and cloned in the expression vector pRSET A. The bacteria BL21 were transformed with the plasmid LmPPDK/pRSET. The protein has been found to be soluble and has been purified. Currently drug screening is being conducted to analyze potential drug compounds that will target leishmania major. In conclusion, the PPDK purified protein will allow us to analyze potential inhibitors that will disable Leishmania major.