Agitoxin-2isapotentandselectiveblockeroftheShakertypevoltage-gated Kv1.3 andKv1.1channels.Agitoxin-2inhibitsKv1.3withanIC50 valueofaround200pMandKv1.1withanIC50 valueofaround140pM.ThispeptidetoxinwasoriginallyisolatedfromthevenomoftheIsraeliscorpion L.quinquestriatushebraeus.Description:Productcode:N/A.Categories:Kv1.3channel,Potassiumchannels.Tags:168147-41-9,Kv1.1,Kv1.3.AAsequence: Gly-Val-Pro-Ile-Asn-Val-Ser-Cys8-Thr-Gly-Ser-Pro-Gln-Cys14-Ile-Lys-Pro-Cys18-Lys-Asp-Ala-Gly-Met-Arg-Phe-Gly-Lys-Cys28-Met-Asn-Arg-Lys-Cys33-His-Cys35-Thr-Pro-Lys-OHDisulfidebonds: Cys8-Cys28;Cys14-Cys33;Cys18-Cys35Length(aa): 38Formula: C169H278N54O48S8MolecularWeight: 4090.89DaAppearance: WhitelyophilizedsolidSolubility: waterandsalinebufferCASnumber: 168147-41-9Source: SyntheticPurityrate: >97%Reference:RecombinantExpressionofMargatoxinandAgitoxin-2inPichiapastoris:AnEfficientMethodforproductionofKV1.3TheK(v)1.3voltage-gatedpotassiumchannelregulatesmembranepotentialandcalciumsignalinginhumaneffectormemoryTcellsthatarekeymediatorsofautoimmunediseasessuchasmultiplesclerosis,type1diabetes,andrheumatoidarthritis.Thus,subtype-specificK(v)1.3blockershavepotentialfortreatmentofautoimmunediseases.SeveralK(v)1.3channelblockershavebeencharacterizedfromscorpionvenom,allofwhichhaveanα/βscaffoldstABIlizedby3-4intramoleculardisulfidebridges.Chemicalsynthesisiscommonlyusedforproducingthesedisulfide-richpeptidesbutthisapproachistimeconsumingandnotcosteffectiveforproductionofmutants,fusionproteins,fluorescentlytaggedtoxins,orisotopicallylabelledpeptidesforNMRstudies.RecombinantproductionofK(v)1.3blockersinthecytoplasmofE.coligenerallynecessitatesoxidativerefoldingofthepeptidesinordertoformtheirnativedisulfidearchitecture.AnalternativeapproachthatavoidstheneedforrefoldingisexpressionofpeptidesintheperiplasmofE.colibutthisoftenproduceslowyields.Thus,wedevelopedanefficientPichiapastorisexpressionsystemforproductionofK(v)1.3blockersusingmargatoxin(MgTx)andagitoxin-2(AgTx2)asprototypicexamples.ThePichiasystemenabledthesetoxinstobeobtainedinhighyield(12-18mg/L).NMRexperimentsrevealedthattherecombinanttoxinsadopttheirnativefoldwithouttheneedforrefolding,andelectrophysiologicalrecordingsdemonstratedthattheyarealmostequipotentwiththenativetoxinsinblockingK(V)1.3(IC(50)valuesof201±39pMand97±3pMforrecombinantAgTx2andMgTx,respectively).FurThermore,bothrecombinanttoxinsinhibitedT-lymphocyteproliferation.AMgTxmutantinwhichthekeypharmacophoreresidueK28wasmutatedtoalaninewasineffectiveatblockingK(V)1.3anditfailedtoinhibitT-lymphocyteproliferation.Thus,theapproachdescribedhereprovidesanefficientmethodofproducingtoxinmutantswithaviewtoengineeringK(v)1.3blockerswiththerapeuticpotential.AnangiR., etal.(2012)RecombinantExpressionofMargatoxinandAgitoxin-2inPichiapastoris:AnEfficientMethodforproductionofKV1.3ChannelBlockers. PLoSONE.PMID:23300835 Purificationandcharacterizationofthreeinhibitorsofvoltage-dependentK+channelsfromLeiurusquinquestriatusvar.hebraeusvenomThreenewtoxinsfromthevenomofthescorpionLeiurusquinquestriatusvar.hebraeushavebeenidentifiedonthebasisoftheirabilitytoblocktheShakerK+channel.ThesetoxinshavebeenpurifiedusingHPLCtechniquesandcharacterizedas38aminoacidpeptidesbymassspectroscopy,aminoacidanalysis,andsequencedetermination.Theirchemicalidentitywasconfirmedbyproducingfullyfunctionalsynthetictoxinsusingrecombinantmethods.ThesepeptidesarepotentinhibitorsoftheShakerK+channel(Kd<1nM)aswellasthemammalianhomologuesofShaker.TheyarerelatedtootherpreviouslydescribedK+channeltoxins,butformanewsubclasswithinthelargerfamilyofK+channelinhibitorsderivedfromscorpionvenom.Wehavenamedthesetoxinsagitoxin1,2,and3,respectively.Garcia,M.L. etal.(1994)Purificationandcharacterizationofthreeinhibitorsofvoltage-dependentK+channelsfromLeiurusquinquestriatusvar.hebraeusvenom. Biochemistry.PMID:8204618