CLONING, EXPRESSION ANALYSIS AND HETEROLOGOUS PRODUCTION OF SNAKE VENOM VASCULAR ENDOTHELIAL GROWTH FACTORS (svVEGF) FROM THREE BOTHROPIC SNAKE SPECIES

Junqueira-de-Azevedo, I.L.M.; Silva, M.B.; Farsky, S.H.P.; Ho, P.L.

Centro de Biotecnologia, Instituto Butantan, São Paulo, SP

The analysis of abundant Expressed Sequence Tags (ESTs) from the Viperidae snake Bothrops insularis venom glands revealed the occurrence of 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 similarities with vertebrate VEGFs, placental growth factors (PlGF), platelet-derived growth factor (PDGF) and with the hypotensive factor (HF) from Vipera aspis venom. To investigate its possible role in the snake venoms, the cDNA was subcloned, expressed in E. coli with a 6X His-tag as an insoluble monomer and was purified by a Ni2+-affinity chromatography after 8M urea extraction. Antiserum against the recombinant svVEGF was generated in mice and tested in Western-blot against proteins from various snake venoms and cellular extracts of snake venom glands and other tissues, showing that the mature svVEGF is ubiquitously distributed throughout snake venoms, even from Elapidae family, but not expressed in other cellular extracts. This result was also confirmed by Northern Blot studies of RNAs from other related Viperidae species and by cloning  and complete sequencing of svVEGFcDNAs from B. jararaca and B. erytromelas pitviper. The recombinant protein was also dimerized after refolding processes and biologically characterized, showing ability to increase vascular permeability. These results indicate 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 [J. Biol. Chem. (2001) 276 (43) pp 39.836].

Supported by FAPESP, CNPq and Fundação Butantan.

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Inácio de Loiola Meirelles Junqueira de Azevedo, Rua Dr. Queiróz Guimarães, 640, São Paulo, SP, CEP: 05609-000, Brasil, Email: ijuncaze@usp.br