Under the supervision of Amber Eubanks, a third-year graduate student and I have been studying the biochemistry of kinases in Plasmodium, the parasite that causes malaria. Malaria kills hundreds of thousands of people each year across the globe. The primary classes of patients affected are pregnant women and children under the age of five and primarily affects Africa, Southeast Asia and South America. It is already known that kinases make excellent drug targets in Plasmodium because they are often unique and many do not have human host homologs. This means that I can specifically target parasite kinases over human kinases. Compounds that fit this profile will make viable drug candidates because they will not have off-target effects in human patients during treatment.
In addition to making excellent drug targets, kinases are involved in many of the critical biological processes within organisms, however, the roles of many kinases in Plasmodium are unknown. In fact, over 40% of the parasite genome has unpredicted functions at present. Our aim is to elucidate their functions/roles within the parasite to better understand the Plasmodium biology, develop probes for further studying the parasites and also to lend more information about pathways that can be targeted in the parasites.
More specifically, we are working with PfPK9 and its substrate, UBC13 (ubiquitin-conjugating enzyme 13) which are very specific to the Plasmodium species. Essentially, the goal is to test whether PfPK9 phosphorylates UBC13 since previous experiments have shown that phosphorylation of UBC13 negatively regulates its activity (Haystead). We are currently working with IPTG which mimics allo-lactose to turn on the lac-operon in order to begin translation of DNA. However, since IPTG is only a mimic, it cannot be hydrolyzed by and Beta-galoctosidase therefore the concentration of IPTG stays the same and this leads to more production of PfPK9.
Fractions show the gradient
Additionally, we use protein purification methods such as batch or column binding with glutathione to link the GST-PfPK9 to a sepharose resin in order to separate using affinity. Collecting the fractions that have the highest peaks indicate the most probable locations of PfPK9. I then perform gel electrophoresis and Western blots to see whether PfPK9 has been successfully isolated from contaminants in order to then be able to begin a hydrolysis assay.
Derbyshire Lab material
Although isolating one kinase seems nearly impossible, PfPK9 has no known homologs therefore we can use gel filtration or an anion exchange column which separates the positively charged protein from the negatively charged sepharose resin in order to isolate PfPK9. This is a very general overview of my project in the lab and I hope to be able to create a stronger foundation within research as I advance in my chemistry and biology courses at Duke and with additional lab experience
