RESUMO
PnTx3-1 is a peptide isolated from the venom of the spider Phoneutria nigriventer that specifically inhibits A-type K(+) currents (I(A)) in GH(3) cells. Here we used a bacterial expression system to produce an NH(2)-extended mutant of PnTx3-1 (ISEF-PnTx3-1) and tested whether the toxin is functional. The recombinant toxin was purified from bacterial extracts by a combination of affinity and ion-exchange chromatography. The recombinant toxin blocked A-type K(+) currents in GH(3) cells in a fashion similar to that observed with the wild-type toxin purified from the spider venom. These results suggest that recombinant cDNA methods provide a novel source for the production of functional Phoneutria toxins. The recombinant ISEF-PnTx3-1 should be useful for further understanding of the role of A-type K(+) currents in biological processes.
Assuntos
Neuropeptídeos/biossíntese , Bloqueadores dos Canais de Potássio , Proteínas Recombinantes de Fusão/biossíntese , Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Fracionamento Químico , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , Dados de Sequência Molecular , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Venenos de Aranha/farmacologia , Células Tumorais CultivadasRESUMO
We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10 percent of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K