Expression and purification of RNA Pseudouridylate synthase putative of malaria parasite (Plasmodium falciparum)

Abstract

The high morbidity and mortality from malaria infection, especially amongst children under five years of age, necessitates the production of new antimalarial drugs. Plasmodium falciparum RNA pseudouridylate synthase putative (PfRPuSP) is an essential enzyme in protein synthesis, making it a potential drug target. Therefore, this study investigated the cloning, transformation, expression and purification of PfRPuSP. Both (pET28a (+) and pGEX-6P-1) plasmids were utilised for the cloning of PfRPuSP. The heat shock standard method was used for the gene constructs transformation in three strains of E. coli. Terrific and Luria Broth media at 25 °C and 30 °C were utilised to optimise the growth conditions. Nickel-nitrilotriacetate resin and fast protein liquid chromatography were employed for PfRPuSP purification to obtain its pure form. In this study, the best expression of PfRPuSP was achieved using PF3D7_1227900_ pET28a(+) construct in C41(DE3) competent cells overnight at 25 °C in LB medium as depicted by the Western blot results. The PF3D7_1227900_ pGEX-6P-1 construct yielded no desired result after expression and purification. The best medium, buffer, E.coli strain and matrix resin were determined, which can be used as baseline information for further studies and monitoring. These discoveries would be highly valuable in uncovering a way to form and understand the three-dimensional structure of PfRPuSP. This knowledge could lead to the development of medications that can block the enzyme in Plasmodium falciparum, ultimately aiding in eradicating malaria parasites.