Expressão heteróloga de neurotoxinas do veneno da aranha Phoneutrianigriventer

• Ano de defesa: 2011
• Autor(a) principal: Ivana Assis Souza
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Phoneutria nigriventer; Glutamato; Toxinas; Tx3-4 Tx3-6; Canais de cálcio; Expressão heteróloga; Bloqueadores de canais de cálcio; Ácido glutamico; Toxinas biológicas; Venenos de aranha; Canais de calcio; Neurotoxinas
• Link de acesso: http://hdl.handle.net/1843/BUOS-8M7GCR

The venom of the spider Phoneutria nigriventer contains several peptides that act on ion channels and receptors of the nervous system of insects and mammals. The toxins Tx3-6 and Tx3-4 are voltage-gated calcium channel blockers and have shown a pharmacological potential in the treatment of pain and in neuroprotection. Only small amounts of these toxins can be purified from the venom and pharmacological and structural tests depend on their availability. In this work, we tested different expression and purification systems in order to identify the best method of getting functional toxins Tx3-6 and Tx3-4 in large amounts. The recombinant toxin Tx3-6 expressed in the vector pBADmycHis in E. coli was functional, however, the expression yield was only 0,3 mg/L of culture. Two miligrams of Tx3-6 was obtained using the SUMO system (E. coli) but the recombinant toxin was not functional. The Tx3-4 was also expressed in the SUMO system and approximately 800 g/L of a soluble functional recombinant toxin was produced. Three miligrams of insoluble Tx3-4 was also purified by the SUMO system and after refolding in vitro, we got 2 mg/L of a functional toxin. Expressions carried in the systems pPICZ (yeast), pET (E. coli) and pBADHis (E. coli) were unsuccessful due to low yield, expression as inclusion bodies and production of inative toxins. The secondary structural caracterization of the recombinant toxin Tx3-4 by circular dichroism revealed that the peptide is mainly arranged in random coil but it has some -helices and, in a lower percentage, -sheet structures are also present. This structure is similar to that described for the oxytoxins from the venom of the spider Oxyopes lineatus, which are also voltage-gated calcium channel blockers. Despitethe availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. The functional expression of the toxins Tx3-6 and Tx3-4 created new expectations for further pharmacological and structural analysis and the results obtained from the refolding of the Tx3-4 opened new pathways for the production of these toxins in large scale