Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs.
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.
Loyevsky
Mark
M
Center for Sickle Cell Disease, Howard University, 2121 Georgia Avenue, NW, Washington, DC 20059, USA. mloyevsky@howard.edu
Mompoint
Farah
F
Yikilmaz
Emine
E
Altschul
Stephen F
SF
Madden
Thomas
T
Wootton
John C
JC
Kurantsin-Mills
Joseph
J
Kassim
Olakunle O
OO
Gordeuk
Victor R
VR
Rouault
Tracey A
TA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D006801:Humans; D035925:Iron-Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010963:Plasmodium falciparum / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D051153:Wnt Proteins; D029961:Zebrafish Proteins
D017931:DNA Primers; D035925:Iron-Regulatory Proteins; D011518:Proto-Oncogene Proteins; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D011505:Protein-Tyrosine Kinases