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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME228MembraneMembraneProteinProteinProtocolsProtocolsExpression,Purification,Expression,Purification,andCharacterizationandCharacterizationEditedbyEditedbyBarryS.SelinskyBarryS.Selinsky1ExpressionandPurificationoftheAmphipa
2、thicFormofRabbitCytochromebinEscherichiacoli5LucyWaskell1.IntroductionCytochromeb(cytb)isanelectrontransferproteinthatexistsina55membrane-boundformintheendoplasmicreticulumwhereitisanchoredtothemembraneviaacarboxyl-terminaltransmembrane-helix(1–3).Themembrane-boundformofcytbpr
3、ovidesreducingequivalentsforthebiosyn-5thesisofavarietyoflipidsincludingunsaturatedfattyacids,plasmalogens,andcholesterol.Inaddition,itfacilitatesthecytochromeP450catalyzedoxidationofselectedsubstrates(2).Themembranedomainislinkedtotheamino-terminalcatalyticheme-containingdomai
4、nviaan11aminoacidlinker.Themammaliancytsbaretypicallygreaterthan90%similarinsequenceandmay5beinterchangeableinsomesystems(4).Nevertheless,ourlaboratoryusesrab-bitcytochromeP4502B4andcytochromeP450reductaseandhaselectedtousetherabbitcytbsoallproteinsarefromasinglespecies.Cytbals
5、oexists55inasolubleforminredbloodcellswhereitfunctionstomaintainhemoglobininitsferrousoxygen-carryingform(5).2.Materials2.1.Escherichiacoli(E.coli)Strains,Media,andEquipment1.ThekeytothemarkedandreproducibleoverexpressionofthemembraneboundformofcytbisuseoftheE.colistrainC41,ade
6、rivativeofE.coliDE3(Avidis5SA,SaintBeauzire,FR)(6).Thisstrainmoderatestheexpressionofgenesdown-streamfromaT7promoter,thereby,decreasingthetoxicityofthelargeamountofmRNAgenerated(7)(LifeTechnologies-GibcoBRL)(seeNote1).From:MethodsinMolecularBiology,vol.228:MembraneProteinProtoc
7、ols:Expression,Purification,andCharacterizationEditedbyB.S.Selinsky©HumanaPressInc.,Totowa,NJ34Waskell2.LBAgar,32g/Lwater.Autoclaveat121°Cfor15min.Coolandpourintoplates.Addcarbenicillintoafinalconcentrationof100g/mL(LifeTechnologies-GibcoRBL).3.LuriaBertani(LB)medium:10g/LNaCl,1
8、0g/Lpeptone,5g/Lyeastextract,1mL1NNaOH.4.Terrificbroth(
