EXPRESSION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANT PALLIDIPIN, A NOVEL PLATELET-AGGREGATION INHIBITOR FROM THE HAEMATOPHAGEOUS TRIATOMINE BUG TRIATOMA-PALLIDIPENNIS

Pallidipin is a platelet aggregation inhibitor protein originating fro m the saliva of the haematophageous triatomine bug Triatoma pallidipen nis. Its inhibitory effects are specific for collagen-induced platelet aggregation. The recombinant form of the protein was expressed in the periplasmic space of transformed Escherichia coli using a vector base d on the alkaline phosphatase gene promoter and leader peptide. Recomb inant pallidipin was purified in three chromatographic steps including cation exchange, anion exchange and size exclusion gel chromatography . SDS/PAGE and N-terminal amino acid sequencing showed that recombinan t pallidipin had a molecular weight similar to that of the salivary pr otein (similar to 19 kDa) and had been correctly processed. The yield was 864 mu g of pure protein from one litre of bacterial culture. The biological activity of recombinant pallidipin was assessed in a platel et aggregation assay using collagen at a concentration of 2 mu g/ml as inducer, and the IC50 found to be 33 nM, similar to that determined f or the native protein. When the collagen concentration used for induct ion was increased, higher pallidipin concentrations were also needed t o achieve a comparable inhibition of platelet aggregation.