title: Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties.
abstract: Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni(2+)-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 × 10(8) min(-1) M(-1), 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
authors.forename: Balamurugan
authors.initials: B
authors.lastname: Sundaram
authors.forename: Nandan Mysore
authors.initials: NM
authors.lastname: Varadarajan
authors.forename: Pradeep Annamalai
authors.initials: PA
authors.lastname: Subramani
authors.forename: Susanta Kumar
authors.initials: SK
authors.lastname: Ghosh
authors.forename: Viswanathan Arun
authors.initials: VA
authors.lastname: Nagaraj
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D000913:Antibodies, Protozoan / Q000276:immunology / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006072:Gossypol / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010072:Oxamic Acid / Q000378:metabolism; D010966:Plasmodium vivax / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D007770:L-Lactate Dehydrogenase; D006072:Gossypol; D010072:Oxamic Acid