Expression, Optimization of Purification and Molecular Modeling of a Disintegrin-like protein from Agkistrodon contortrix laticinctus

Ribeiro, J.U.; Ramos, O.H.P.; Selistre-de-Araújo, H.S.

Depto. de Ciências Fisiológicas, Universidade Federal de São Carlos, SP.

Disintegrins are proteins found in some snake venoms. Due to their potential to inhibit the integrin functions, these proteins have been used in the study of many phenomena of adhesion. Disintegrins having the RGD sequence are capable to inhibit the number of experimental of metastasis and to prolong the survival of guinea pigs without toxic effect. On the other hand, little is known about the potential of the non-RGD disintegrins. The cDNA coding for ACLD-C, a non-RGD disintegrin, was cloned based in the sequence of its precursor metaloprotease/disintegrin ACLD (GenBank U86634), obtained from a cDNA library of venom gland from the snake Agkistrodon contortrix laticinctus. The molecular modeling of ACLD-C suggests that there is no interaction of this molecule with integrin aVb3, as it was found for RGD disintegrins. It was developed an expression system to ACLD-C in fusion with MBP (maltose binding protein) in E. coli, using pMalD vector. ACLD-C/MBP fusion protein was purified through affinity chromatography in an amilose column and by gel-filtration chromatography in a Superdex-200 column. Cell adhesion assays are in progress for the study of interaction of this recombinant protein with integrins expressed in normal and tumor cells.

Financial support: CNPq, FAPESP.

Correspondence to: julianauema@yahoo.com