Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from Plasmodium knowlesi for the Development of Anti-Malarial Drugs

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.

Keywords: Plasmodium knowlesi; lactate dehydrogenase; malaria; protein expression; protein purification.

Figure 1

Digestion of PCR product (Pk-LDH gene) and expression vector pET21a with NheI and XhoI. Red arrows showing A1 and A2 lanes were the digested PCR product (Pk-LDH gene). Green arrows showing V1 and V2 lanes were the digested pET21a expression vector.

Figure 2

SDS-PAGE analysis showing the expression level and the solubility of Pk-LDH cultured in different broths. Overexpressed protein can be seen when using TB broth with added MgSO4 (lane A3). M: stained molecular weight markers in kDa (Protein ladder). Lane 1: LB Broth, Lane 2: M9 broth, Lane 3: Terrific broth, Lane 4: ZYP broth.

Figure 3

The elution profile of Pk-LDH protein showed by gel filtration using SuperdexTM 200 GL 10/300 column, which exhibited a single peak protein elution (1) (blue trace). Brown trace indicates salt solution (2).

Figure 4

The purified protein exhibited a single band at 34 kDa based on 12% SDS-PAGE analysis. kDa: Protein Ladder, Lane 1: Crude Protein, Lane 2: Unbound Protein after IMAC, Lane 3: Eluted Protein after IMAC, and Lane 4: Eluted Protein after size exclusion chromatography.

Figure 5

Mascot score histogram and protein sequence coverage of Pk-LDH. (a) Mascot Score Histogram of Pk-LDH. Ions score is −10 × Log (p), where p is the probability that the observed match is a random event. Individual protein scores > 45 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. (b) Protein sequence coverage of Pk-LDH compared with lactate dehydrohenase from P. knowlesi (AGL33703.1). Matched peptides shown in red bold.

Figure 6

Diagram of CD spectrometry far-UV analysis. Two typical negative peaks can be seen at 211 nm and 222 nm.