Identification, cloning, and mutational analysis of the casein kinase 1 cDNA of the malaria parasite, Plasmodium falciparum. Stage-specific expression of the gene.
The cDNA for casein kinase 1 (CK1) of Plasmodium falciparum was cloned, sequenced, and expressed in bacteria. The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of approximately 37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms. The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1-7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP. A casein kinase activity, partially purified from soluble extracts of P. falciparum by affinity chromatography through CK1-7 columns displayed identical properties. The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring >> schizont. Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite. Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme. The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.
Barik
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Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama 36688, USA. sbarik@jaguar1.usouthal.edu
Taylor
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Chakrabarti
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D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D047388:Casein Kinases; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases