Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi.

Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, Plasmodium ovale morphologically resembles Plasmodium vivax, resulting in misidentification and confusion in diagnosis, and is responsible for malarial disease relapse due to the formation of hypnozoites. P. ovale receives relatively less attention compared to other major parasites, such as P. falciparum and P. vivax, primarily due to its lower pathogenicity, mortality rates, and prevalence rates. To efficiently produce lactate dehydrogenase (LDH), a major target for diagnosing malaria, this study used three Escherichia coli strains, BL21(DE3), BL21(DE3)pLysS, and Rosetta(DE3), commonly used for recombinant protein production. These strains were characterized to select the optimal strain for P. ovale LDH (PoLDH) production. Gene cloning for recombinant PoLDH production and transformation of the three strains for protein expression were performed. The optimal PoLDH overexpression and washing buffer conditions in nickel-based affinity chromatography were established to ensure high-purity PoLDH. The yields of PoLDH expressed by the three strains were as follows: BL21(DE3), 7.6 mg/L; BL21(DE3)pLysS, 7.4 mg/L; and Rosetta(DE3), 9.5 mg/L. These findings are expected to be highly useful for PoLDH-specific diagnosis and development of antimalarial therapeutics.