title: Enhanced purification and characterization of the PfeIF4A (PfH45) helicase from Plasmodium falciparum using a codon-optimised clone.
abstract: With the intention of investigating the DNA strand displacement properties of Plasmodium falciparum helicase PfeIF4A (formerly known as PfH45) a codon-optimized gene for expression in Escherichia coli has been produced. Several histidine-containing proteins with intrinsic helicase activity were captured from the bacterial sonicate by initial Ni(2+)-chromatography. Heparin and size-exclusion steps were subsequently required for unambiguous PfeIF4A purification. This strategy generated an active recombinant protein of significantly improved yield in comparison to previously published studies (~4.2 mg/g wet weight of cells). Helicase unwinding assays confirmed a bipolar activity, but revealed a preference for unwinding a free 3'-end, with a rate of displacement in the 3'-5' direction 2-fold higher than that in the 5'-3' direction. DNA constructs with two, three or four blunt ends were not unwound. Studies confirmed the enzyme to be Mg(2+)-dependent, optimally active at 37°C and had a background ATP turnover rate of 23.16±1.74 pmol/min, which in the presence of single- or double-stranded DNA doubled to 42.92±3.21 pmol/min.
authors: IBBS Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, Hampshire, United Kingdom.
authors: Luke
authors: L
authors: Evans
authors: Darren
authors: D
authors: Gowers
authors: Keith
authors: K
authors: Firman
authors: James
authors: J
authors: Youell
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D016366:Open Reading Frames; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D004247:DNA; D039601:Eukaryotic Initiation Factor-4A; D000251:Adenosine Triphosphatases; D008274:Magnesium