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 Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate 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 insoluble monomer, and purified by Ni(2+)-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.
Junqueira de Azevedo
I L
IL
Centro de Biotecnologia, Laboratório de Imunoquimica, Instituto Butantan, 1500 São Paulo 05503-900, Brazil.
Farsky
S H
SH
Oliveira
M L
ML
Ho
P L
PL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017837:Bothrops / Q000235:genetics*; D002199:Capillary Permeability; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000096:biosynthesis / Q000235:genetics*; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D011994:Recombinant Proteins / Q000096:biosynthesis; D030162:Reptilian Proteins / Q000096:biosynthesis / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D003435:Crotalid Venoms; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; D030162:Reptilian Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors