Cell-penetratingpeptideandionchannelsblocker
Crotamine isa basicpeptide presentinthe venom ofthe SouthAmerican rattlesnakeCrotalusdurissusterrificus.MultipleBIOLOGicalfunctionshavebeenattributedtoCrotamine.Itisanaturalcell-penetratingpeptidewithselectivebiologicalactiontowardsactivelyproliferatingcelltypes.Moreover,ithasbeenreportedthatcrotamineisablockerofKv1.3(IC50around300nM)aswellasKv1.1andKv1.2.Ithasanalgesicpropertiesandmyonecroticeffects.Inaddition,crotaminebelongstothebeta-defensinpeptidesandassuchdemonstratesantibacterialpropertiesbyinteractingwithlipidmembranes.
Description:
AAsequence: YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG-OH
Disulfidebridges: Cys4-Cys36;Cys11-Cys30;Cys18-Cys37
Length(aa):41
Formula:C214H326N64O54S7
MolecularWeight:4883.82Da
Appearance:whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:
Source:synthetic
Purityrate:>95%
Reference:
CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus
DeLuccaFL.,etal.(1974)CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus(Ophidia,Reptilia)aftermanualextractionofthevenom.Studiesontemplateactivityandbasecomposition.BiochemJ.PMID:4463939
Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus
Crotamine,a4.88kDaneurotoxicprotein,hasbeenpurifiedtoapparenthomogeneityfromCrotalusdurissusvenombygelfiltrationonSephadexG-75.Wheninjected(i.p.ors.c.)inadultmaleSwissmice(20-25g),itinducedatime-dosedependentanalgesiceffectwhichwasinhibitedbynaloxone,thussuggestinganopioidactionmechanism.Whencomparedwithmorphine(4mg/kg),crotamine,eveninextremelylowdoses(133.4microg/kg,i.p.,about0.4%ofaLD50isapproximately30-foldmorepotentthanmorphine(w/w)asananalgesic.Onamolarbasisitismorethan500-foldmorepotentthanmorphine.Itisalsomuchmorepotentthanthelowermolecularweightcrudefractionsofthesamevenom.Theantinociceptiveeffectsofcrotamineandmorphinewereassayedbythehotplatetestandbytheaceticacid-inducedwrithingmethod.Therefore,bothcentralandperipheralmechanismsshouldbeinvolved.Histopathologicalanalysisofthebrain,liver,skeletalmuscles,stomach,lungs,spleen,heart,kidneysandsmallintestineofthecrotamineinjectedmicedidnotshowanyvisiblelesioninanyoftheseorgansbylightmicroscopy.Sincecrotamineaccountedfor22%(w/w)ofthedesiccatedvenom,itwasidentifiedasitsmajorantinociceptivelowmolecularweightpeptidecomponent.
MancinAC.,etal.(1998) Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus(SouthAmericanrattlesnake)venom:abiochemicalandpharmacologicalstudy.Toxicon.PMID:9839677
Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus
Hereinwereportthatcrotamine,asmalllysine-andcysteine-richproteinfromthevenomoftheSouthAmericanrattlesnake,canrapidlypenetrateintodifferentcelltypesandmouseblastocystsinvitro.Invivocrotaminestronglylabelscellsfrommousebonemarrowandspleenandfromperitonealliquid,asshownbyfluorescentconfocallaser-scanningmicroscopy.Nuclearlocalizationofcrotaminewasobservedinbothfixedandunfixedcells.Inthecytoplasm,crotaminespecificallyassociateswithcentrosomesandthusallowsustofollowtheprocessofcentrioleduplicationandseparation.Inthenucleus,itbindstothechromosomesatS/G2phase,whencentriolesstartdividing.Moreover,crotamineappearsasaMarkerofactivelyproliferatingcells,asshownby5-BrdUcell-proliferationassay.CrotamineinthemicromolarrangeprovednontoxictoanyofthecellculturestestedanddidnotaffectthepluripotencyofEScellsorthedevelopmentofmouseembryos.
KerkisA.,etal.(2004)Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus.FASEBJ.PMID:15231729
BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake
Moleculesisolatedfromanimals,insects,plantsormicroorganismscanprovideprototypesfordesignofbiopharmaceuticalproducts.Somevenomtoxinsandtheirderivativesareusedinmedicine,whileothersprovidetemplatesfordevelopmentofnewdrugs.Themildtoxin,crotamine,asmallbasiclow-molecular-weightpolypeptidepurifiedfromthevenomofaSouthAmericanrattlesnake,Crotalusdurissusterrificus.Crotaminewasdiscoveredmorethan50yearsagoandonlyinthepastsixyearshasitsexceptionalbiologicalversatilitybeendemonstrated.Particularly,itscell-penetratingABIlity,whichallowscrotaminetocrosscellmembranesandtoaccumulateinthenucleus;itsuseforintracellularvesicletrackingandasacellcyclemarkeranditscapabilityfordeliveringDNAintoreplicatingmammaliancells.Bothantimicrobialactionandpotentialselectiveantitumoractivityofcrotaminehavealsobeenfound.Multidisciplinaryapproachesandpathwaysofdiscoveryplacedcrotamineinararecategoryofversatilebiomolecules,inwhichconcentration,moleculartargetpreference,structuralancestryandspecificitytowardbiologicalmembranesplayanintegralrole.Crotamineisadruggablepeptidewithhighpotentialforuseasanimagingagentfordetectingdividingcells,forintracellulardeliveryofhydrophilicbiomolecules,andasanalternativechemotherapeuticcompoundagainstaggressivetypesofcancer.
KerkisI.,etal.(2010)BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake.ExpertOpinInvestigDrugs.PMID:21062230
Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake
Animalvenomscompriseanaturallyselectedcocktailofbioactivepeptides/proteinsandothermolecules,eachofwhichplayingadefinedrolethankstothehighlyspecificinteractionswithdiversemoleculartargetsfoundintheprey.Researchfocusedonisolation,structural,andfunctionalcharacterizationsofnovelnaturalbiologics(bioactivepeptides/proteinsfromnaturalsources)hasalongwaytogothroughfromthebasicsciencetoclinicalapplications.Herein,weoverviewthestructuralandfunctionalcharacteristicsofthemyoneurotoxincrotamine,firstlyisolatedfromtheSouthAmericanrattlesnakevenom.Crotamineisthefirstvenompeptideclassifiedasanaturalcellpenetratingandantimicrobialpeptide(CPPandAMP)withamorepronouncedantifungalactivity.IncontrasttootherknownnaturalCPPsandAMPs,crotaminedemonstratesawidespectrumofbiologicalactivitieswithpotentialbiotechnologicalandtherapeuticvalues.Morerecentstudieshavedemonstratedtheselectiveinvitroanticanceractivityofcrotamine.Invivo,usingamurinemelanomamodel,itwasshownthatcrotaminedelaystumorimplantation,inhibitstumorcellsproliferation,andalsoincreasesthesurvivalofmiceengraftedwithsubcutaneousmelanoma.Thestructuralandfunctionalpropertiesandalsothepossiblebiotechnologicalapplicationsofminimizedmoleculesderivedfromcrotaminearealsodiscussed.
KerkisI.,etal.(2014)Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake.BiomedResInt.PMID:24551848
Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.
Ourgoalwastodemonstratetheinvivotumorspecificaccumulationofcrotamine,anaturalpeptidefromthevenomoftheSouthAmericanrattlesnakeCrotalusdurissusterrificus,whichhasbeencharacterizedbyourgroupasacellpenetratingpeptidewithahighspecificityforactivelyproliferatingcellsandwithaconcentration-dependentcytotoxiceffect.Crotaminecytotoxicityhasbeenshowntobedependentonthedisruptionoflysosomesandsubsequentactivationofintracellularproteases.Inthiswork,weshowthatthecytotoxiceffectofcrotaminealsoinvolvesrapidintracellularcalciumreleaseandlossofmitochondrialmembranepotentialasobservedinrealtimebyconfocalmicroscopy.Theintracellularcalciumoverloadinducedbycrotaminewasalmostcompletelyblockedbythapsigargin.Microfluorimetryassaysconfirmedtheimportanceofinternalorganelles,suchaslysosomesandtheendoplasmicreticulum,ascontributorsfortheintracellularcalciumincrease,aswellastheextracellularmedium.Finally,wedemonstrateherethatcrotamineinjectedintraperitoneallycanefficientlytargetremotesubcutaneoustumorsengraftedinnudemice,asdemonstratedbyanoninvasiveopticalimagingprocedurethatpermitsinvivoreal-timemonitoringofcrotamineuptakeintotumortissue.Takentogether,ourdataindicatethatthecytotoxicpeptidecrotaminecanbeusedpotentiallyforadualpurpose:totargetanddetectgrowingtumortissuesandtoselectivelytriggertumorcelldeath.
NascimentoFD(2012)Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.MolPharm.PMID:22142367
Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.
Crotamineisabasic,42-residuepolypeptidederivedfromsnakevenomthathasbeenshowntopossesscell-penetratingproperties.CrotamineformsnanoparticleswithavarietyofDNAandRNAmolecules,andcrotamine-plasmidDNAnanoparticlesareselectivelydeliveredintoactivelyproliferatingcellsincultureorinmice.Assuch,thesenanoparticlescouldformthebasisforanucleicaciddrug-deliverysystem.Herewedescribethepreparation,purification,andbiochemicalandbiophysicalanalysisofvenom-derived,recombinant,chemicallysynthesized,andfluorescent-labeledcrotamine;theformationandcharacterizationofcrotamine-DNAand-RNAnanoparticles;andthedeliveryofthesenanoparticlesintocellsandanimals.
HayashiMA.,etal.(2012)Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.MethodsMolBiol.PMID:22791447
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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!
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
两个CEX方法A和B测定同一单抗,结果碱性峰比例差不多,酸性峰比例相差约7%,相应主峰也差了7%左右。
具体来说,A方法酸性峰高,主峰低,碱性峰稍微低点;B方法酸性峰低,主峰高,碱性峰稍微高点;另外也做了CIEF,结果呢和A方法更接近。
仔细比较起来,AB两个方法的峰性和数量差不多,就不知道为什么会有这么大的差异。两个方法一个用的WCX柱-磷酸缓冲液,一个用SCX柱-MES缓冲液
大家帮我分析下:
1.两个方法哪个方法更准确,是以酸性峰高的为准还是什么?为什么?
2.这显著差异是由方法造成,具体原因是什么?柱子?
3.CIEF的结果和A方法更接近,是不是可以由此证明A方法更好或者CIEF的方法更好(因为CIEF更快更方便)?
欢迎讨论~
纠正下,A方法用的是Tosoh的柱子,B方法用的是SCX柱。TOSOH的柱子是7um的填料,10cm长。SCX是10um的填料。我本人TOSOH的阳离子柱子用的很少,这次信手用用,结果发现差异很大
那我现在就考虑,在以后方法开发过程中,除了通过流动相pH和组成、梯度、柱子选择来获得样品主峰和酸碱性的最大分离,还要关注各峰比例。因为之前比较方法好坏都只看分离度,尤其是主峰和邻近峰的分离度,获得最大分离度,自然可以做到主峰尽可能纯,但从未认真比较过各峰比例。这是一个大疏忽吧!
另外,CIEF和CEX方法原理还是有点差异的,所以分的是不同的异质体,原液放行两个方法肯定是都要做的。问题就是在早期细胞株筛选和工艺开发阶段,哪个方法才是又快又准。CIEF(iCE280)一般15分钟一个样,比CEX快多了。如果CIEF测得主峰要低于CEX结果,是不是真的完全可以取代CEX呢?CEX分离出的峰远比CIEF的多!
欢迎大家继续讨论~
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。
这就是说不用酸碱预处理吗?
Whatman的网站上没有DE52最大耐受压力,请问又经验的战友应该是多少?
Whatman的网站上:
DE32DryMicrogranularDEAECellulose
SimilarperformancecharacteristicsafterprecyclingasDE52.
DE52PreswollenMicrogranularDEAECellulose
ProbablythemostwidelyusedDEAEcelluloseintheworld;usedforbiopolymerswithlowtohighnegativecharges;exhibitsexcellentresolutionwithgoodflowrates.
附件是一本图书(MethodsinMolecularMedicine,)的章节,上面说:
WhatmanDEAE52comesalreadypreswollenandonlyneedstobetransferred
totherunningbuffer50mMTE8.
lAntibodiesUsingIonExchangeChromatography.pdf(87.06k)
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