Enhanced purification and characterization of the PfeIF4A (PfH45) helicase from Plasmodium falciparum using a...

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.