RESUMO
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 ß-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% ß 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.
Assuntos
Antimaláricos/metabolismo , L-Lactato Desidrogenase/metabolismo , Malária/metabolismo , Plasmodium knowlesi/enzimologia , Antimaláricos/química , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/química , Malária/terapiaRESUMO
Production of ATP by the glycolytic pathway in the mammalian pathogenic stage of protists from the genus Trypanosoma is required for the survival of the parasites. Cofactor-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shows no similarity to the corresponding enzyme in humans, and has also been genetically validated as a target by RNAi experiments. It has previously been shown that trypanosomatid iPGAMs require Co(2+) to reach maximal activity, but the biologically relevant metal has remained unclear. In this paper the metal content in the cytosol of procyclic and bloodstream-form T. brucei (analysed by inductively coupled plasma-optical emission spectroscopy) shows that Mg(2+), Zn(2+) and Fe(2+) were the most abundant, whereas Co(2+) was below the limit of detection (<0.035 µM). The low concentration indicates that Co(2+) is unlikely to be the biologically relevant metal, but that instead, Mg(2+) and/or Zn(2+) may assume this role. Results from metal analysis of purified Leishmania mexicana iPGAM by inductively coupled plasma-mass spectrometry also show high concentrations of Mg(2+) and Zn(2+), and are consistent with this proposal. Our data suggest that in vivo cellular conditions lacking Co(2+) are unable to support the maximal activity of iPGAM, but instead maintain its activity at a relatively low level by using Mg(2+) and/or Zn(2+). The physiological significance of these observations is being pursued by structural, biochemical and biophysical studies.