Cloning, expression and functional activity of deoxyhypusine synthase from <it>Plasmodium vivax</it>

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible <it>in vitro </it>cultivation method. Sequencing of the <it>Plasmodium vivax </it>genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from <it>P. falciparum</it>, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite <it>P. falciparum</it>. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe <it>Plasmodium </it>species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites.</p> <p>Results</p> <p>We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (<it>dhs</it>) sequence from genomic DNA of <it>P. vivax </it>PEST strain Salvador I (Accession number <ext-link ext-link-type="gen" ext-link-id="AJ549098">AJ549098</ext-link>) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot.</p> <p>The putative DHS protein from <it>P. vivax </it>displays a FASTA score of 75 relative to DHS from rodent malaria parasite, <it>P. yoelii</it>, and 74 relative to that from the human parasite, <it>P. falciparum </it>strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in <it>E. coli </it>BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from <it>P. vivax</it>, despite an amino acid identity of 44% between the proteins.</p> <p>Conclusion</p> <p>We identify a novel DHS protein in the more benign malaria parasite,<it>P. vivax</it>, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, <it>P. yoelii</it>, and human <it>P. falciparum </it>strains.</p>