Transmembrane localization of LdNT2

Nucleoside transporters play a critical role in successful intracellular parasitism by Leishmania donovani. The current drugs available for treatment of leishmaniasis are highly toxic due to their lack of specificity for the parasite. The aim of this research project is the biochemical and functional characterization of the LdNT2 transporter of Leishmania donovani. It is only through a better understanding of the nutritionally essential purine transporters that these proteins can be validated as potential therapeutic targets and be exploited to ferry conventional and experimental drugs into the parasite. Oligonucleotide primers were constructed to amplify segments of the LdNT2 gene that encode loops between predicted transmembrane domains using the polymerase chain reaction (PCR). The amplified PCR products were subcloned into the TOPO TA vector, their sequences verified, and then cloned into the pGEX-KT vector to synthesize fusion proteins. The fusion proteins will now be purified via affinity chromatography, and then the purified fusion proteins will be used to raise antibodies directed against specific domains of the LdNT2 protein. These antibodies will be employed to immunolocalize and determine the membrane topology of LdNT2. The immunolocalization by electron microscopy and the elucidation of the membrane topology of LdNT2 will provide critical insight into the specialized function of this unique membrane carrier.