title: Cloning, Overexpression, Purification and Immunolocal-ization of PFD0975w from the Malaria Parasite Plasmodium falciparum.
abstract: Malaria is a parasitic disease, widespread along the tropical regions of the world. The disease has killed 4, 38,000 individuals in the year 2015 (WHO). The malaria parasite, Plasmodium falciparum, has evolved resistance to front-line antimalarials over the decade, necessitating the identification of new drug targets. Protein kinases are excellent drug targets since they participate in critical cell-signaling cascades. We have identified a putative RIO-like protein kinase, PFD0975w, from the Plasmodium kinome. It is believed to play a key role in ribosome biogenesis. We have cloned and over-expressed the protein in E. coli and purified it to homogeneity. The recombinant protein is of molecular weight 36.3±1.2 kDa. Purified recombinant PFD0975w is active in vitro and binds ATP. PFD0975w exhibits a unique localization pattern in each RBC stage. PFD0975w localizes within the parasite cytosol during ring stage and spread throughout the infected RBCs during trophozoite and schizont stages with the strongest expression signal during the trophozoite phase indicating the importance of the enzyme in parasite growth and survival. Interestingly, the localization pattern of the protein also responds to stress conditions such as starvation and antimalarial drug pressure. It exhibits punctuate pattern in the treated parasite during trophozoite and schizont stages compared to untreated parasites, indicating some role of the putative kinase in cellular stress handling. Our results indicate PFD0975w is a potential drug target in the malaria parasite and active recombinant PFD0975w can be exploited to identify, validate or design novel inhibitors.
authors.forename: Arnish
authors.initials: A
authors.lastname: Chakraborty
authors.forename: S N
authors.initials: SN
authors.lastname: Balaji
authors.forename: Amogh A
authors.initials: AA
authors.lastname: Sahasrabuddhe
authors.affiliation: Department of Biosciences & Bioengineering, Indian Institute of Technology- Guwahati. Guwahati-781039, Assam, India. vtrivedi@iitg.ernet.in.
authors.forename: Vishal
authors.initials: V
authors.lastname: Trivedi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D003001:Cloning, Molecular; D016503:Drug Delivery Systems; D055808:Drug Discovery; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases