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1、IPTG诱导的原核表达Chapter15,Protocol1ExpressionofClonedGenesinE.coliUsingIPTG-induciblePromotersThisprotocoldescribeshow(1)toclonecloned>Thisprotocoldescribeshow(1)tocloneclonedsequencesencodingopenreadingframesinplasmidscarryingIPTG-induciblepromoters,(2)tooptimizeexpressionoftargetproteinsintran
2、sformantscarryingtheserecombinants,and(3)toscale-upproductionofforeignproteins..CAUTION.RECIPE.MATERIALSBuffersandSolutionsCoomassieBrilliantBluestainorSilverstain.IPTG(1M).1xSDSgel-loadingbuffer..NucleicAcidsandOligonucleotidesGeneorcDNAfragmentofinterest.MediaLBagarplatescontaining50µg/ml
3、ampicillin.LBmediumcontaining50µg/mlampicillin..AdditionalReagentsStep1ofthisprotocolrequiresthereagentslistedinChapter8,Protocol7.Step2ofthisprotocolrequiresthereagentslistedinChapter1,Protocol17orChapter1,Protocol19.Step3ofthisprotocolrequiresthereagentslistedinChapter1,Protocol23toChapte
4、r1,Protocol26.Step4ofthisprotocolrequiresthereagentslistedinChapter12,Protocol3..VectorsandBacterialStrainsE.colistrainsuitablefortransformationandcarryingeitherthelacIqorlacIq1alleleSomeIPTG-inducibleexpressionvectorscarrythelacIqalleleontheexpressionplasmid(e.g.,pMALandpGEX).Thesecanbeuse
5、dinanylaboratorystrainofE.coli(e.g.,JM101,DH5F',andTG1).IPTG-inducibleexpressionvectorOtherexamplesincludepGEM-3Z(Promega),pGEX-1(Pharmacia),pKK223-3(Pharmacia),pMEX(U.S.Biochemicals),pTrc99A(Pharmacia),andpMAL(NewEnglandBiolabs).Positivecontrolplasmid(e.g.,anIPTG-induciblevectorknowntoexpr
6、essaLacZfusionproteinofdefinedsize)..METHOD1..ModifybyPCR(Chapter8,Protocol7),orisolatebyrestrictionenzymedigestion,afragmentofDNAcarrying5'-and3'-restrictionenzymesitescompatiblewithsitesinanIPTG-inducibleexpressionvector.ConstructsgeneratedbyPCRshouldbesequencedtoensurethatnospuriousmutat
7、ionswereintroducedduringtheamplificationreactions.2..LigatetheDNAfragmentcontainingthecDNA/geneofinterestintotheexpressionvector(Chapter1,Protocol17orChapter1,Protocol19).3..TransformanE.colistraincontainingthelacIqallelewiththerecombinantplasmid.Ifthepl
