Expression of a functional recombinant Phoneutria nigriventer toxin active on K+ channels.
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.
Carneiro
A M D
AM
Departamento de Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, Av Antonio Carlos, 6627, Belo Horizonte, 31270-901, Minas Gerais, Brazil.
Kushmerick
C
C
Koenen
J
J
Arndt
M H L
MH
Cordeiro
M N
MN
Chavez-Olortegui
C
C
Diniz
C R
CR
Gomez
M V
MV
Kalapothakis
E
E
Prado
M A M
MA
Prado
V F
VF
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
D009479:Neuropeptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D013111:Spider Venoms; C098736:Tx3 neurotoxin