Abstract
Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl β-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% β strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.
Highlights
Given the high incidence of malaria in many tropical and subtropical areas, it has long been identified as one of the most devastating diseases in the world
The findings indicated that the P. knowlesi lactate dehydrogenase (Pk-LDH) sequence homology of 90.5%, 89.0% and 88.9% with P. falciparum LDH (ABA46355), P. malariae was almost identical to the published sequence of the lactate dehydrogenase of P. knowlesi
It is noteworthy that the search for protein homology homology of 90.5%, 89.0% and 88.9% with P. falciparum LDH (ABA46355), P. malariae LDH, against the NCBI
Summary
Given the high incidence of malaria in many tropical and subtropical areas, it has long been identified as one of the most devastating diseases in the world. Malaria-related deaths occur primarily among children under five years of age, which highlights the need for specific measures to protect this population group from malaria infection [1]. Naturallyacquired human malaria is usually related with four species of Plasmodium: Plasmodium falciparum, P. vivax, P. malariae and P. ovale. Zoonotic malaria infections are increasingly being reported. This new type of infection is caused by P. knowlesi—a species previously known to infect primates. The first naturally human infection was reported in Kapit Division of Sarawak in 2004 [2]. P. knowlesi infection may be catastrophic, because the parasites possess an asexual erythrocytic cycle of about 24 h, leading to a fever that typically occurs at the same frequency [5]. Current treatment for malaria caused by P
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