原核表达中文版pET操作手册

pETSystemManua[e-mail]889(LIC)9F.pET10T7lac10pLysSpLysE101011pLacI11pETcoco™11G.11H.121212121313pET14I.16J.CE616K.17II.18A.pET18B.19C.20D.21E.22III.23A.23B.23232425pETSystemManua[e-mail]™/2626PCR26/PCR28/PCR29293030IV.30A.31B.DE3313131V.32A.323132323333B.34C.33353534pET35pET35D.36N-3636mRNA373737mRNA37VI.38SDS-PAGE383838A.PopCulture™39B.39C.39D.40E.41BugBuster®Benzonase®41rLysozyme™4243F.43BugBuster®4343G.PopCulture™44pETSystemManua[e-mail]-PAGE495B./46VIII.47A.48B.50IX.5050X.51λDE35153pET54pET?54XI.T7•pETNovagen•NovagenMerckKgaAEMD•NovagenpETSystemManua[e-mail]()T7RNAT7RNA50%T7RNA(I.F.)pLpIT7RNACE6lacUV5T7RNAIPTG()T7pETpET/B.NovagenT7T7RNAC.pETpETDNA10µgBL21BL21(DE3)BL21(DE3)pLysS1,23pET100.2mlNovaBlueBL21(DE3)BL21(DE3)pLysSSOC1pET31BLRBLR(DE3)pLysSBL212pETTrx32AD494BL21NovagenNovagen()pETD.pETpETStudier(StudierandMoffatt,1986;Rosenbergetal.,1987;Studieretal.,1990)NovagenpET•AUG3pET-21(+)pET-24(+)pET-23(+)•T7pET(7)pET-21a(+)BamHIGGATCCaGGAbGATcBamHIATCdcNcoINdeIAUG18pETSystemManua[e-mail]•••pET----pETE.coli(pET7N)(LIC)Xa(pET7)pETPCRLICPCR/1)[-S-(GST)(Trx)NusA(NutilizationsubstanceA)]2)(TrxDsbADsbC)3)(trxBgor12)DsbADsbCpET-39b(+)pET-40b(+)/pETDsbADsbC(7)1)2)pET-17xbN220aaT710pET-31b(+)S•Tag™T7•Tag®GST•Tag™His•Tag®HSV•Tag®Nus•Tag™WesternBlot()His•TagGST•TagS•TagT7•TagpETSystemManua[e-mail]•TagGST•TagFRETWorks™S•Tag1fmolHis•TagCBD•Tag™[pET-34b(+)35b(+)CBDclos•Tag]CBDsCBinDTMCBD•TagNus•Tag™Trx•Tag™GST•Tag™Nus•TagTrx•TagOrigami™OrigamiBRosetta-gami™(12)pETpET5'(7)5'(Xa)C-(1)(2)pETN/C(I)()/pETT7•Tag®N,I11260AP,IF,IP,WB3,5,9,11,1717x,21,23,24,28,33S•Tag™N,I15S-(104aa)AP,QA,WB29,30,32,34–37,39–44His•Tag®N,C,I6,8,10()AP,IF,WB14–16,19–44HSV•Tag®C11IF,WB25,27,43.1,44pelB/ompTN20/22PE12,20,22,25,26,27KSIN125PP31Trx•Tag™N109trxBtrxB/gorDB,SP32PKAsiteI5APS33CBDclos•TagN156IP,AP,WB34,35CBDcenA•TagN114/IP,AP,PE,WB36,37CBDcex•TagC107/IP.AP,PE,WB38Dsb•Tag™N208(DsbA)236(DsbC)DB,DI,PE,SP39,40GST•Tag™N220AP,IF,IP,QA,WB41,42Nus•Tag™N,I495SP,WB43.1,44AP=IP=QA=DB=PE=SP=DI=PP=/WB=WesternIF=PS=pETSystemManua[e-mail](+)f1DNAkanRT7lacT7•Tag®11STag™CBDTag™HSV•Tag®Dsb•Tag™NusTag™ampRT7His•Tag®T7Tag260TrxTag™KSIPKAGST•Tag™pET-3a-dNpET-9a-dNpET-11a-dNpET-12a-cpET-14bNTpET-15bNTpET-16bNXpET-17bNpET-17xbNpET-19bNEpET-20b(+)CpET-21a-d(+)CNpET-22b(+)CpET-23a-d(+)CNpET-24a-d(+)CNpET-25b(+)CCpET-26b(+)CpET-27b(+)CCpET-28a-c(+)N,CITpET-29a-c(+)CNTpET-30a-c(+)N,CIT,EpET-30Ek/LICN,CIT,EpET-30Xa/LICN,CIT,XpET-31b(+)CNpET-32a-c(+)I,CINT,EpET-32Ek/LICI,CINT,EpET-32Xa/LICI,CINT,XpET-33b(+)N,CIITpET-34b(+)CINT,EpET-35b(+)CINT,XpET-36b(+)CINT,EpET-37b(+)CINT,XpET-38b(+)CICTpET-39b(+)I,CINT,EpET-40b(+)I,CINT,EpET-41a-c(+)I,CINT,EpET-41Ek/LICI,CINT,EpET-42a-c(+)I,CINT,XpET-43.1a-c(+)I,CICNT,EpET-43.1Ek/LICI,CICNT,EpET-44a-c(+)N,I,CICIT,EkanRT7lacT7•Tag11S•TagCBDTagHSV•TagDsb•TagNusTagampRT7His•TagT7•Tag260Trx•TagKSIPKAGST•TagT7Tag11=11aaT7Tag260=260aasignalseq.=I=N=N-C=C-T=E=X=XapETSystemManua[e-mail]=E.pETDNApET(LIC)LICLICLICpET(-)pETT7IPTG-(Mr31.5kDa)T7RNApETpETNdeINcoI5'-AUGC-pETNcoI(CCATGG)ATGT7RNAN-AUGPCRNcoINcoI[BspHI(TCATGA)BspLU11I(ACATGT)AflIII(ACRYGT)StyI(CCWWGG)]NcoI()()(pET7)BbsI(BpiI,BpuAI)5’-GAAGAC(N)2–3’3’-CTTCTG(N)6-5’GAAGACNNNNNNNCTTCTGNNNNNNNBsaI(Eco31I)5’-GGTCTC(N)1-3’3’-CCAGAG(N)6-5’GGTCTCNNNNNNCCAGAGNNNNNNBsmBI(Esp3I)5’-CGTCTC(N)1-3’3’-GCAGAG(N)5-5’CGTCTCNNNNNNGCAGAGNNNNNNBspMI5’-ACCTGC(N)4–3’3’-TGGACG(N)8-5’ACCTGCNNNNNNNNNTGGACGNNNNNNNNNPCRNcoI4pETSystemManua[e-mail]•Tag™[pET-41a-c(+)42a-c(+)]Nus•Tag™[pET-43.1a-c(+)pET-44a-c(+)]XaPshAISmaI()Xa23(Chang1985LeBonniec1991LeBonniec1996)(LIC)LICPCR(AslanidisanddeJong1990Haunetal.1992)LICpET12–155'LICT4DNA3'?5'pETLICXaLICTB163TB205pETSystemManual[e-mail]“”E.coliDE3pETT7/T7lacpLysSpLysET7lacT7lac(Studieretal.1990DubendorffandStudier19917)T7laclac(lacI)T7laclacIDE3laclacUV5T7RNAT7lacT7RNABL21(DE3)HMS174(DE3)(DubendorffandStudier1991)pLysSpLysE()pLysSpLysET7T7RNA(MoffattandStudier1987Studier1991)T7(Inouyeetal.1973)T7RNA(Zh