title: Expression of a functional recombinant Phoneutria nigriventer toxin active on K+ channels.
abstract: 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.
authors.affiliation: Departamento de Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, Av Antonio Carlos, 6627, Belo Horizonte, 31270-901, Minas Gerais, Brazil.
authors.forename: A M D
authors.initials: AM
authors.lastname: Carneiro
authors.forename: C
authors.initials: C
authors.lastname: Kushmerick
authors.forename: J
authors.initials: J
authors.lastname: Koenen
authors.forename: M H L
authors.initials: MH
authors.lastname: Arndt
authors.forename: M N
authors.initials: MN
authors.lastname: Cordeiro
authors.forename: C
authors.initials: C
authors.lastname: Chavez-Olortegui
authors.forename: C R
authors.initials: CR
authors.lastname: Diniz
authors.forename: M V
authors.initials: MV
authors.lastname: Gomez
authors.forename: E
authors.initials: E
authors.lastname: Kalapothakis
authors.forename: M A M
authors.initials: MA
authors.lastname: Prado
authors.forename: V F
authors.initials: VF
authors.lastname: Prado
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013111:Spider Venoms / Q000235:genetics* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D009479:Neuropeptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D013111:Spider Venoms; C098736:Tx3 neurotoxin