ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF TRANSFERRIN FROM THE TSETSE FLY, GLOSSINA MORSITAN CENTRALIS

Heamatophagous insects such as tsetse flies face an oxidative stress due to blood meal from vertebrate hosts. Iron binding proteins such as transferrin are therefore crucial for their survival by limiting iron toxicity. Mid gut homogenate and heamolymph samples were screened for iron binding proteins by staining of proteins with iron-binding protein specific stain, Ferene-S. Polyacrylamide gel electrophoresis (PAGE) under denaturing conditions showed the presence of iron binding proteins in the low (Mr~30 kDa) and high (Mr~66 kDa) molecular weight ranges. Native PAGE indicated the presence of two iron binding protein bands of molecular weights 490 kDa and 140 kDa. Heamolymph samples were subjected to KBr density gradient ultracentrifugation (206,000xg, 4h, 4°C) to remove the major heamolymph protein, lipophorin. The putative transferrin was electroeluted from the gel following separation of the subphase through a preparative non-denaturing PAGE. Further purification was carried out through affinity chromatography using concanavalin-A Sepharose column. The putative transferrin band was bound to the column indicating glycosylation with mannose rich carbohydrate residues. Isolated protein was further analysed through SDS-PAGE and two-dimensional gel electrophoresis.