15-E.coli Expression Systems

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1、MolecularBiologyProblemSolver:ALaboratoryGuide.EditedbyAlanS.GersteinCopyright©2001byWiley-Liss,Inc.ISBNs:0-471-37972-7(Paper);0-471-22390-5(Electronic)15E.coliExpressionSystemsPeterA.BellExpressionVectorStructure............................462WhatMakesaPlasmidanExpressionVector?...........462

2、IsaStrongerPromoterAlwaysDesirable?..............463WhyDoPromotersLeakandWhatCanYouDoaboutIt?.........................................464WhatFactorsAffecttheLevelofTranslation?............464WhatCanAffecttheStabilityoftheProteinintheCell?.........................................464WhichProtein

3、ExpressionSystemSuitsYourNeeds?.......465TrackRecord.......................................465WhatDoYouKnowabouttheGenetoBeExpressed?...465WhatDoYouKnowaboutYourProtein?...............468AdvertisementsforCommercialExpressionVectorsAreVeryPromising.WhatLevelsofExpressionShouldYouExpect?........

4、..............................470WhichE.coliStrainWillProvideMaximalExpressionforYourClone?......................................471WhyShouldYouSelectaFusionSystem?...............471WhenShouldYouAvoidaFusionSystem?...............472IsItNecessarytoCleavetheTagofftheFusionProtein?...............

5、...........................474WillExtraAminoAcidResiduesAffectYourProteinofInterestafterDigestion?............................475WorkingwithExpressionSystems.......................475WhatAretheOptionsforCloningaGeneforExpression?.......................................475461IsScreeningNecessary

6、PriortoExpression?............476WhatAspectsofGrowthandInductionAreCriticaltoSuccess?..........................................478WhatAretheOptionsforLysingCells?................479Troubleshooting.......................................480NoExpressionoftheProtein.........................480TheP

7、roteinIsExpressed,butItIsNottheExpectedSizeBasedonElectrophoreticAnalysis...................480TheProteinIsInsoluble,NowWhat?..................481SolubilityIsEssential.WhatAreYourOptions?...........482TheProteinIsMade,butVeryLittleIsFul