RECOMBINANT TOXINS FROM Triatoma infestans SALIVARY GLAND - MOLECULAR CLONING AND THREE DIMENSIONAL STRUCTURE

Feijó G.(1,2) Martins, N.F.(3), Santana, J.M.(1), Lozzi, S.P.(1), Teixeira, A.R.L.(1)

(1)Laboratório Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasília. (2)Universidade Católica de Brasília, (3)Laboratório de Bioinformática, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil.

Saliva of bloodsuckers contains various regulators of some hemostatic stages. Some saliva proteins activate the host’s fibrinolytic system (Basanova et all, 2002). The understanding of these proteins structure and functions could lead to a feasible mean to control insect-borne diseases. For this purpose we have carried on studies on biochemical, molecular purification and modeling of salivary proteins from the kissing bug Triatoma infestans. Therefore, a cDNA library was constructed in the l ZAP phage, and rabbit antibodies were used for immune screening. Three gene sequences were selected and transferred to high expression plasmids where recombinant proteins were obtained in prokaryotic cells: Triatin, Infestilin and triatox have been obtained in sufficient amounts. Preliminary tests show hemorrhagic activity in vitro. The sequences were submitted to bioinformatics analysis and molecular modeling prediction. A similarity search performed with BLASTp showed low identity with nitrophorin and others salivary gland proteins for the three genes. Against the protein data bank the BLAST search selected the thrombin inhibitor form T. pallidipenis as a possible template for molecular modeling with 30% identity between the template and the targets. The threading structure prediction techniques for homology modeling revealed the scaffold similar to the lipocalinfamily. Lipocalins are remarkably diverse at the sequence and function level yet have highly conserved structures. The conserved motifs fold in highly symmetrical all-b structure dominated by a single eighth stranded antiparallel beta-strand flattened in its elliptical shape. This work present the three dimensional structure of the three proteins and discuss the structure-function relationship.

CORRESPONDENCE TO:

Natália Florêncio Martins, Rua 9 Norte Bloco 4 ap. 1202, Aguas Claras, Brasilia, DF, CEP: 70000000, Brasil, Email: natalia@cenargen.embrapa.br