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

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...

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Autores principales:	Nurhainis Ogu Salim, Fazia Adyani Ahmad Fuad, Farahayu Khairuddin, Wan Mohd Khairulikhsan Wan Seman, Mohd Anuar Jonet
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	lactate dehydrogenase malaria <i>Plasmodium knowlesi</i> protein expression protein purification Organic chemistry QD241-441
Acceso en línea:	https://doaj.org/article/358ce8e7ebae4496a21a8555d5ce7602
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spelling	oai:doaj.org-article:358ce8e7ebae4496a21a8555d5ce76022021-11-11T18:35:49ZPurifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs10.3390/molecules262166251420-3049https://doaj.org/article/358ce8e7ebae4496a21a8555d5ce76022021-11-01T00:00:00Zhttps://www.mdpi.com/1420-3049/26/21/6625https://doaj.org/toc/1420-3049Plasmodium 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 <i>Plasmodium knowlesi</i> in a bacterial system. Synthetic <i>P. knowlesi</i> lactate dehydrogenase (<i>Pk</i>-LDH) gene was cloned into pET21a expression vector, transformed into <i>Escherichia coli</i> 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 <i>Pk</i>-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 <i>P. knowlesi</i> 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 <i>Pk</i>-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble <i>Pk</i>-LDH that is biologically active was produced, which can be used further in other malaria studies.Nurhainis Ogu SalimFazia Adyani Ahmad FuadFarahayu KhairuddinWan Mohd Khairulikhsan Wan SemanMohd Anuar JonetMDPI AGarticlelactate dehydrogenasemalaria<i>Plasmodium knowlesi</i>protein expressionprotein purificationOrganic chemistryQD241-441ENMolecules, Vol 26, Iss 6625, p 6625 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	lactate dehydrogenase malaria <i>Plasmodium knowlesi</i> protein expression protein purification Organic chemistry QD241-441
spellingShingle	lactate dehydrogenase malaria <i>Plasmodium knowlesi</i> protein expression protein purification Organic chemistry QD241-441 Nurhainis Ogu Salim Fazia Adyani Ahmad Fuad Farahayu Khairuddin Wan Mohd Khairulikhsan Wan Seman Mohd Anuar Jonet Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs
description	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 <i>Plasmodium knowlesi</i> in a bacterial system. Synthetic <i>P. knowlesi</i> lactate dehydrogenase (<i>Pk</i>-LDH) gene was cloned into pET21a expression vector, transformed into <i>Escherichia coli</i> 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 <i>Pk</i>-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 <i>P. knowlesi</i> 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 <i>Pk</i>-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble <i>Pk</i>-LDH that is biologically active was produced, which can be used further in other malaria studies.
format	article
author	Nurhainis Ogu Salim Fazia Adyani Ahmad Fuad Farahayu Khairuddin Wan Mohd Khairulikhsan Wan Seman Mohd Anuar Jonet
author_facet	Nurhainis Ogu Salim Fazia Adyani Ahmad Fuad Farahayu Khairuddin Wan Mohd Khairulikhsan Wan Seman Mohd Anuar Jonet
author_sort	Nurhainis Ogu Salim
title	Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs
title_short	Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs
title_full	Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs
title_fullStr	Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs
title_full_unstemmed	Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs
title_sort	purifying and characterizing bacterially expressed soluble lactate dehydrogenase from <i>plasmodium knowlesi</i> for the development of anti-malarial drugs
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/358ce8e7ebae4496a21a8555d5ce7602
work_keys_str_mv	AT nurhainisogusalim purifyingandcharacterizingbacteriallyexpressedsolublelactatedehydrogenasefromiplasmodiumknowlesiiforthedevelopmentofantimalarialdrugs AT faziaadyaniahmadfuad purifyingandcharacterizingbacteriallyexpressedsolublelactatedehydrogenasefromiplasmodiumknowlesiiforthedevelopmentofantimalarialdrugs AT farahayukhairuddin purifyingandcharacterizingbacteriallyexpressedsolublelactatedehydrogenasefromiplasmodiumknowlesiiforthedevelopmentofantimalarialdrugs AT wanmohdkhairulikhsanwanseman purifyingandcharacterizingbacteriallyexpressedsolublelactatedehydrogenasefromiplasmodiumknowlesiiforthedevelopmentofantimalarialdrugs AT mohdanuarjonet purifyingandcharacterizingbacteriallyexpressedsolublelactatedehydrogenasefromiplasmodiumknowlesiiforthedevelopmentofantimalarialdrugs
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Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from <i>Plasmodium knowlesi</i> for the Development of Anti-Malarial Drugs

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