PMID:12615322
| Citation |
Loyevsky, M, Mompoint, F, Yikilmaz, E, Altschul, SF, Madden, T, Wootton, JC, Kurantsin-Mills, J, Kassim, OO, Gordeuk, VR and Rouault, TA (2003) Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs. Mol. Biochem. Parasitol. 126:231-8 |
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| Abstract |
Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa. |
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| Keywords |
Animals; Base Sequence; Binding Sites; DNA Primers; Humans; Iron-Regulatory Proteins/biosynthesis; Iron-Regulatory Proteins/genetics; Iron-Regulatory Proteins/metabolism; Jurkat Cells; Molecular Sequence Data; Nucleic Acid Conformation; Plasmodium falciparum/metabolism; Protein Binding; Protein-Tyrosine Kinases/metabolism; Proto-Oncogene Proteins/metabolism; Protozoan Proteins/biosynthesis; Protozoan Proteins/genetics; Protozoan Proteins/metabolism; RNA, Protozoan/chemistry; RNA, Protozoan/genetics; Recombinant Proteins/metabolism; Wnt Proteins; Zebrafish Proteins |
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