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1、CP20160205A-PKRwt-ECITaskTopurifythePKRwtproteinsfromE.colisystem2Procedure2.1BufferLysisbufferA:50mMTris,5()0mMNaCl,pH&()IBwashbufferB:50mMTris,500mMNaCl,1%TritonX-100,600mMGuanidine-HCl,pH8.0NiSepharoseFFcolumn32ml(GE,XK26,6cm)BindingBufferC:50mMTris,6MGuanidi
2、ne-HCl,1OmMimidazole,pH8.0ElutionBufferD:50mMTris,6MGuanidine-HCl,500mMimidazole,pH&ORefoldingbufferE:50mMTris,pH8.0,0」MKC1,2O%glycerol,5mMEDTA,lmMDTT,0JMNBSD201DialysisbufferF:50mMTris,lOOmMKC1,20%Glycerol,pH8.02.2ExpressionBL21(DE3)pET28a-PKRwta.Inoculate10()m
3、lLB(Kana)with100ulglycerolstocks(37°Cx200rpm)overnight;b.Add20mlovernightcultureinto2LLBmedia(Kana)(37°Cx200rpm)untilOD600reaches0.8;c.AddIPTGtoafinalconcentrationof0.5mMtoinduceproteinexpression;e.Continuegrowing(37°Cx200rpm)4h,f.Centrifugeat6,000gfor20minat4°C
4、,collectcellpellet,1Lharvest5g.2.3Micro-fluidicslysatea.Resuspend5gcellspelletin50mllysisbufferA,b.Lysedwiththepressure12,000psi,3times,c.Centrifugelysateat40,000gfor20minat4°C,collectpellets.2.4IBwasha.Resuspendthepelletsin50mLIBwashbufferB,b.Centrifugeat6,000g
5、for10minat10°C,collectpellet,c.Repeata-btwiced.Resuspendthepelletsin50mLlysisbufferAe.Centrifugeat6,000gfor10minat10°C,collectpellet,f.Repeatd-eonceagain,g.Resuspendthepelletsin50mLbufferCbystirredovernight.2.5NiSepharoseFFcolumna.Centrifugelysateat40,000gfor20m
6、inat4°C,collectsupernatant,a.filterwith0.45umb.loadthesupernatantontothepre-equilibratedNiSepharoseFFcolumnc.WashthecolumnwithbufferCuntilnoproteinwasdetectedinflowthroughd.Washthecolumnwith4%bufferDuntilnoproteinwasdetectedinflowthroughe.Elutethecolumnwith50%an
7、d100%bufferDuntilnoproteinwasdetectedinflowthroughf.Run12%SDS-PAGE.2.1refoldinga.collected50%elution(30ml)andDTTwasaddedto25mM,b.dilutedto500mlrefoldingbufferEbydropwise,c.incubateat4°Covernight.2.2concentratedanddialysisa.Centrifugeat20000gfor20minat4°Candcolle
8、ctsupernatantb.Concentratedfrom500mLto30mlusingVivaflow200MWCO30,000c.Centrifugeat20000gfor20minat4°Candcollectsupernatantd.DialysisagainstthestoragebufferFovernight2
