Optimisation of purification of plasmodium falciparum Kelch13 protein for structural analysis

Abstract

The protozoan Plasmodium falciparum has the potential to cause severe malaria disease which is fatal when untreated. Artemisinin derived from Artemisia annua L. has conventionally been used to combat the disease. However, resistance to Artemisinins originating from Southeast Asia has been developing, compromising the fight against malaria. Though Artemisinins is believed to target widespread cellular targets, resistance phenotype has been traced to mutations in the Plasmodium falciparum Kelch13 protein, which is essential for intraerythrocytic growth of the parasite. Unfortunately, the development of new drugs has been impeded by the challenge to express and purify the Kelch13 protein. In this study, strategies to optimise the expression and purification of the Kelch13 protein by modulating cell growth and extraction conditions were proposed. Techniques such as SDS-PAGE, size exclusion chromatography, and negative-stain transmission electron microscopy verified the presence of soluble proteins when low temperature, high nutrient conditions were used in combination with a detergent, reducing agent and high salt buffers. Negative-stain transmission electron microscopy images displayed a largely monomeric structure of the Kelch13 protein in solution. The results obtained in this study proves the ability of the Kelch13 to be expressed in solution as a monomer, empowering future studies in antimalarial drug development.