cDNA Cloning, Heterologous Expression, Cytotoxicity, and Inhibitory Effects of a Disintegrin from Bothrops ammodytoides Venom

Abstract

AbstractAn mRNA transcript that codes for a Disintegrin and a Metalloprotease from a venom gland of the viper Bothrops ammodytoides was isolated. Vector pCR®2.1-TOPO was used to clone the Disintegrin transcript and then subcloned in the pET-28a vector to express the protein in the E. coli strain BL21. The recombinant disintegrin, HisrDisintegrin, comprises 38 residues at the N-terminal and 75 residues of the Disintegrin, including 14 cysteines or 7 disulfide bonds. HisrDisintegrin was obtained soluble in the intracellular fraction and separated by affinity chromatography. The experimental molecular mass of HisrDisintegrin, 11,750.8 Da, agreed with its theoretical value, including the cleavage of a Met residue caused by a bacterial post-translational modification. HisrDisintegrin was folded in in vitro conditions and then purified, finding a main fraction with a comparable molecular mass. The HisrDisintegrin contains an alpha/beta structure, as observed by circular dichroism. Its biological activity demonstrated the growth reduction of the human endothelial (HMEC-1) cells and the human mammary gland adenocarcinoma (MDA-MB-231). Furthermore, HisrDisintegrin inhibits the adhesion of HMEC-1 and MDA-MB-231 cells to the proteins of the extracellular matrix (laminin (LN), fibronectin (FN), and vitronectin (VN)).