Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper - A new member of the VEGF family of proteins

During the generation of abundant expressed sequence tags from the Viperida e snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-l ike) protein. The deduced primary sequence, after complete sequencing of th e longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebr ate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insol uble monomer, and purified by Ni2+-affinity chromatography after 8 m urea e xtraction. Antiserum against svVEGF was generated and tested in Western blo t against proteins from snake venoms and cellular extracts. The mature svVE GF appears to be ubiquitously distributed throughout snake venoms and was a lso confirmed by Northern blot studies of other related Viperidae species a nd by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biological ly characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin durin g the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.