表达质粒启动子分类

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1、表达质粒启动子分类.txt这是一个禁忌相继崩溃的时代，没人拦得着你，只有你自己拦着自己，你的禁忌越多成就就越少。自卑有多种档次，最高档次的自卑表现为吹嘘自己干什么都是天才。ExpressionvectorsGeneexpressionfromforeignpromoters.Manyproteinsareexpressedatlowlevelsinvivo.Toproducehighlevelsofaprotein,itisoftenusefultoclonethegenedownstreamofawell-characterized,regulatedpromoter.Inducingtra

2、nscriptionfromtheregulatedpromoterthusresultsinelevatedexpressionofthedownstreamgeneproduct.Iftheregulatedpromotercanbeturnedofftightly,thisalsoprovidesamethodofconditionallydepletingthecellofageneproduct.Avarietyofregulatedpromoterscanbeusedforthispurpose.Afewexamplesaredescribedbelow.PtacThetacpro

3、moter/operator(PTAC)isoneofthemostwidelyusedexpressionsystems.Ptacisastronghybridpromotercomposedofthe-35regionofthetrppromoterandthe-10regionofthelacUV5promoter/operator.ExpressionofPtacisrepressedbytheLaclprotein.ThelacLqalleleisapromotermutationthatincreasestheintracellularconcentrationofLacIrepr

4、essor,resultinginstrongrepressionofPTAC.AdditionoftheinducerlPTGinactivatestheLaclrepressor.Thus,theamountofexpressionfromPTACisproportionaltotheconcentrationoflPTGadded:lowconcentrationsoflPTGresultinrelativelylowexpressionfromPTACandhighconcentrationsoflPTGresultinhighexpressionfromPTAC.Byvaryingt

5、helPTGconcentrationtheamountofgeneproductcloneddownstreamfromPTACcanbevariedoverseveralordersofmagnitude.SeveralpotentialproblemsmustbeconsideredwhenexpressingaclonedgeneproductfromPTAC.lacIAqshouldbeclonedonthesameplasmidastheregulatedgene,becauseiflacIAqisonthechromosomeoronanotherplasmidtheremayb

6、einsufficientLaclproteintofullyrepressthePtacpromoterintrans.ThecellviabilityshouldbemeasuredatdifferentconcentrationsofIPTG,becauseexcessiveoverexpressionofaDNA-bindingproteinmaycausetheproteintoaccumulateininclusionbodies(NilssonandAnderson,1991)orinhibitcellgrowth.Evenwhenfullyrepressed,thereisso

7、meresidualexpressionfromPTAC.Ifthisleakyexpressioncausesproblems,itmaybenecessarytoclonethegeneintoanalternativeexpressionvectorthatismoretightlyrepressed.PBAD.ThepromoterfortheE.coliarabinoseoperon(PBADorPARA)isausefulalternativetoPTAC.WhenageneisclonedbehindthePBADpromoter,expressionofthegeneiscon

8、trolledbytheAraCactivator.ExpressionfromPARAisinducedtohighlevelsonmediacontainingarabinose.Moreover,expressionfromPARAistightlyshutoffonmediacontainingglucosebutlackingarabinose.(ForanexampleofapBADexpressionsystem,seethepBADvectorsmarketedbyInvitrogen.)Likethearabinoseoperon,expressionoftheE.colir

9、hamnoseoperonistightlyregulatedbyanactivator.Expressionfromtherhamnosepromoter(PRHA)isinducedtohighlevelsbytheadditionofrhamnose.Phagepromoters.Anotherapproachthatiswidelyusedforproteinoverexpressionistoplaceageneunderthecontrolofaregulatedphagepromoter.Agenemaybecloneddownstreamofatightlyregulatedp

10、hagepromoterthatisnormallytranscribedbythehostsRNApolymerase.Forexample,expressionofagenecloneddownstreamofthelambdaPLpromotercanberegulatedbythecIrepressor.UsingthetemperaturesensitivecI857repressorallowscontrolofgeneexpressionbychangingthegrowthtemperature-at30CthecI857repressorisfunctionalandittu

11、rnsoffexpressionofthegene,butat42Ctherepressorisinactivatedsoexpressionofthegeneensues.Alternatively,thewild-typecIgenecanbeplacedunderthecontrolofanotherregulatedpromotersuchasthePLACpromoter,allowingregulationbytheadditionofIPTG.(ForanexampleofalambdaPLexpressionsystem,seethePLvectorsmarketedbyInv

12、itrogen.)Alternatively,agenemaybecloneddownstreamofaphagepromoterthatreliesonaphageencodedRNApolymerase.ManyphageproduceaspecificRNApolymerasethatrecognizesapromotersequencewhichisquitedifferentfromE.colipromotersequences.Threephage-specificRNApolymerase/promotersystemsthatarecommonlyoftentoxictothe

13、hostcell.ToavoidpotentialtoxicitythephageRNApolymeraseisonlyinducedwhentheoverexpressionisdesired.Forexample,thephageRNApolymerasemaybeitselfclonedbehindaregulatedpromoter,orthepolymerasemayusedinexpressionvectorsincludeT7,SP6,promoters,thesesystemsresultinveryhighgene.Suchhigh-leveltranscriptioncan

14、productsclonedbehindthephagepromoter,andT3.Inadditiontorecognizinguniquelevelsoftranscriptionofthedownstreambeveryusefulforoverproducinggenebuttheexpressionissohighthatitisbeintroducedtothecellonadefectivephage.(ForexampleofaT7expressionsystem,seethepETvectorsmarketedbyNovagen.)REFERENCES:AmannE,Bro

15、siusJ,PtashneM.1983.Vectorsbearingahybridtrp-lacpromoterusefulforregulatedexpressionofclonedgenesinEscherichiacoli.Gene25:167-178.GuzmanLM,BelinD,CarsonMJ,BeckwithJ.1992.Tightregulation,modulation,andhigh-levelexpressionbyvectorscontainingthearabinosePBADpromoter.JBacteriol.177:4121-4130.Haldimann,A

16、.,L.Daniels,B.Wanner.1998.UseofnewmethodsforconstructionoftightlyregulatedarabinoseandrhamnosepromoterfusionsinstudiesoftheEscherichiacoliphosphateregulon.J.Bacteriol.180:1277-1286.NilssonB,AndersonS.1991.Properandimproperfoldingofproteinsinthecellularenvironment.AnnuRevMicrobiol.45:607-635.todirectStudier,F.,andB.Moffatt.1986.UseofbacteriophageT7RNApolymeraseselectivehigh-levelexpressionofclonedgenes.J.Mol.Biol.189:113-130