Agitoxin-2isapotentandselectiveblockeroftheShakertypevoltage-gated Kv1.3 andKv1.1channels.Agitoxin-2inhibitsKv1.3withanIC50 valueofaround200pMandKv1.1withanIC50 valueofaround140pM.ThispeptidetoxinwasoriginallyisolatedfromthevenomoftheIsraeliscorpion L.quinquestriatushebraeus.
Description:
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-OH
Disulfidebonds: Cys8-Cys28;Cys14-Cys33;Cys18-Cys35
Length(aa): 38
Formula: C169H278N54O48S8
MolecularWeight: 4090.89Da
Appearance: Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: 168147-41-9
Source: Synthetic
Purityrate: >97%
Reference:
RecombinantExpressionofMargatoxinandAgitoxin-2inPichiapastoris:AnEfficientMethodforproductionofKV1.3
TheK(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.hebraeusvenom
ThreenewtoxinsfromthevenomofthescorpionLeiurusquinquestriatusvar.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
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常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
有了源数据之后把源数据按照大小排列，
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
:)
我在做一细菌不同酸碱度生长状况时,发现这些奇怪现象:pH=3的培养基灭菌(TSB液体培养基)灭菌后pH上升到到9.2!而原来pH=9.0的降到8.7(基本没多少变化),请问各位大侠,这是什么原因?
一般做不同酸碱度生长实验时,该如何才能防止pH在湿热灭菌后基本不变化?
是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
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