CLONING AND FUNCTIONAL EXPRESSION OF TX3-2 A LETHAL NEUROTOXIN FROM THE SPIDER Phoneutria nigriventer

Ciscotto, P.H.C., Cordeiro, M.N., Pereira, M.N.A., Diniz C.R., Diniz, M.R.V.

Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil.

Tx3-2, a lethal neurotoxic polypeptide of Mr 3.5 kDa with eight cysteine residues, selectively decreases L-type currents present in GH3 cells. The amino acid sequence homology with toxins of the same venom and from venoms of other spiders which bind with refined specificity to different subtypes of calcium channels, make the Tx3-2 a potential instrument for use in this kind of study. Therefore, a cDNA encoding Tx3-2 was amplified by RT-PCR technique, cloned in pET32c(+) vector DNA, and expressed as a thioredoxin-binding fusion protein in Escherichia coli. After removal of the chaperone protein from the affinity-purified product, the recombinant protein was purified by reverse phase chromatography and shown to be neurotoxicby intracerebral injection in mice. At dose levels of 5 micrograms/mouse, induced immediate clockwise gyration and flaccid paralysis, showing the same symptoms of the native toxin.

Supported by: FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais).

CORRESPONDENCE TO:

DINIZ, M.R.V., Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80. 30510-010. Belo Horizonte (MG), Brazil, Email: mdiniz@funed.mg.gov.br