To further characterize thePlasmodium falciparum cyclic AMP-dependent protein kinase (PfPKA) catalytic subunit, we have cloned and sequenced PfPKA gene. PfPKA sequence shows over 70% homolgy to a previously identified PKA gene from the rodent malaria parasite, P. yoelli. The expression of the PKA catalytic subunit gene in E.coli as either a non-fusion protein or a thioreducin fusion protein has generated a transient expression ofPfPKA however, is prone to rapid degradation during purification steps. Using an alternative system to express the protein as an Intein fusion protein has not resolved the problem of degradation. We're currently attempting to direct the overexpression of PfPKA into inclusion bodies to limit proteolysis in host and also its expression in protease-deficient hosts. We have developed a polyclonal antibody against the C-terminal peptide and the antibody could detect the expressed PfPKA and its degraded byproducts. To examine the effect of cAMP and analogs on P. falciparum, we have incubated malaria parasite with cAMP agonists over an extended period, the parasites have not show any phenotypic change or sexual differentiation. Prolong incubation with PKA inhibitor, H89, could reduce the parasitemia in culture. However, under low concentration of H89, blood stage parasites actually showed elevated PKA activity in the lysate. The mechanism of increased PKA activity remains to be established. To investigate direct effect of PKA in parasite development, we have taken two approaches, one to increase its expression by transient expressionof PfPKA or to disrupt its expression by gene knock-out . Expression plasmid of PfPKA was constructed by using PfPKA sequences and flanking them with the 5' untranslated region (UTR) of P. falciparum histine rich protein 3 (HRP3) and 3' UTR of HRP2. The genetic knock-out clone will be constructed with a modified vector using Taxoplasma gondii DHFR gene as the selection marker.
