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

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.

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