16-Eukaryotic Expression

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1、MolecularBiologyProblemSolver:ALaboratoryGuide.EditedbyAlanS.GersteinCopyright©2001byWiley-Liss,Inc.ISBNs:0-471-37972-7(Paper);0-471-22390-5(Electronic)16EukaryoticExpressionJohnJ.Trill,RobertKirkpatrick,AllanR.Shatzman,andAliceMarcySectionA:APracticalGuidetoEukaryoticExpression.....

2、.492PlanningtheEukaryoticExpressionProject...............493WhatIstheIntendedUseoftheProteinandWhatQuantityIsRequired?........................493WhatDoYouKnowabouttheGeneandtheGeneProduct?.........................................496CanYouObtainthecDNA?..........................497Exp

3、ressionVectorDesignandSubcloning...............498SelectinganAppropriateExpressionHost...............501SelectinganAppropriateExpressionVector.............506ImplementingtheEukaryoticExpressionExperiment.......511MediaRequirements,GeneTransfer,andSelection.......511Scale-upandHarvest

4、................................514GeneExpressionAnalysis.............................515Troubleshooting.......................................517ConfirmSequenceandVectorDesign..................517InvestigateAlternateHosts...........................519ACaseStudyofanExpressedProteinfro

5、mcDNAtoHarvest..........................................519Summary.............................................521SectionB:WorkingwithBaculovirus.....................521PlanningtheBaculovirusExperiment.....................521491IsanInsectCellSystemSuitablefortheExpressionofYourProtei

6、n?.....................................521ShouldYouExpressYourProteininanInsectCellLineorRecombinantBaculovirus?..........................522ProceduresforPreparingRecombinantBaculovirus......524CriteriaforSelectingaTransferVector.................524WhichInsectCellHostIsMostAppropriat

7、eforYourSituation?.........................................525ImplementingtheBaculovirusExperiment................527What’stheBestApproachtoScale-Up?................527WhatSpecialConsiderationsAreThereforExpressingSecretedProteins?.................................527WhatSpecialConsid

8、erationsAreT