An improved fractionation and fast screening method for the identification of new and selective neurotoxins.
Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means of characterizing the growing number of identified ion channels and receptors in the nervous system. Separation procedures leading to the identification of neurotoxins almost always include gel filtration chromatography, combined with ion-exchange and/or reversed phase chromatography. We present here an improved fractionation method based on the use of a new Superdex 30 prep grade HiLoad 16/60 FPLC gel filtration column. This single-step gel filtration protocol results in a shortening of the purification process and allows a superior qualitative separation of (neuro-)peptides in crude venoms as compared to any other type of gel filtration column used thus far. Screening of the collected fractions for potential ion channel blocking properties was performed by means of the whole-cell voltage clamp technique. To increase both the amount and speed of expression in Xenopus laevis oocytes of cloned ion channels, we employed a high-expression vector, pGEMHE, wherein the cDNA encoding a neuronal voltage-dependent potassium channel (RCK1) was subcloned. The combination of these techniques represents a fast and efficient identification procedure in the quest for new and selective neurotoxins for cloned channels and receptors.
Debont
T
T
Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, University of Leuven, Belgium.
Daenens
P
P
Tytgat
J
J
D000818:Animals; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical; D004546:Elapid Venoms / Q000737:chemistry / Q000633:toxicity; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009498:Neurotoxins / Q000302:isolation & purification* / Q000633:toxicity*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects; D012604:Scorpion Venoms / Q000737:chemistry / Q000633:toxicity; D014982:Xenopus laevis
D004546:Elapid Venoms; D009498:Neurotoxins; D015221:Potassium Channels; D012604:Scorpion Venoms