蛋白质分离纯化和复性

90DNA–GMP™™™pH™™™™™•••–scFv57PscFv57PE.Coli–1:10-200010020030040002000400060008000100001200014000Specificactivity(U/mg)Time(min)*200*100*108mol/L6mg/mL10100200SOD020406080100020406080100120140160180200Specificactivity(U/mg)Refoldingtime(h)100µg/ml806040MCAIUU↔I1↔I2↔···↔C↕MAM+M↔AM+I↔AM+C↔AI+C↔A999¾ng/ml)¾2040¾20)–ABABpH02464006008001000120014001600A280NaClDimensionlessvolume(L/(Lmedium))A280(mAU)0.00.10.20.30.4NaCl(mol/L)123456UreapHUrea(mol/L)678910pHabcde0204060Recovery(%)RenaturationmethodSpecificactivityrecoveryActivityrecoveryC0CfC1C2C0C3DenaturedIntermediateWrongfolded•••α•••α1.MingLi,Zhi-GuoSu,andJan-ChristerJanson.Invitroproteinrefoldingbychromatographicprocedures,ProteinExpressionandPurification,33(1):1-10,20042.MingLi,AntonPoliakov,U.HelenaDanielson,ZhiguoSu&Jan-ChristerJanson,Refoldingofrecombinantfull-lengthNS3proteinfromHCVbychromatographicprocedures,BiotechnologyLetters25:1729-1734,20033.MingLiandZhiguoSu,Separationandidentificationofdifferentrefoldingcomponents,J.Biotechnol.,103:119-127,20034.ZhenyuGu,XiaonanZhu,HaimengZhou,andZhiguoSu,Inhibitionofaggregationbymediaselection,sampleloadingandelutioninsizeexclusionchromatographicrefoldingofdenaturedbovinecarbonicanhydraseB,J.Biochem.&Biophys.Methods56(1-3),165-175,20035.M.Li&Z.Su.RefoldinghumanlysozymeproducedasaninclusionbodybyureaconcentrationandpHgradientionexchangechromatography,Chromatographia,56:33-38,20026.M.Li,G.ZhangandZ.Su.Dual-gradientionexchangechromatographyimprovesrefoldingyieldoflysozyme,J.ChromatographyA,959(1-2):113-2020027.M.Li&Z.Su.Refoldingofsuperoxidedismutasebyionexchangechromatography,Biotechnol.Lett.,24:919-923,20028.GuZ,WeidenhauptM,IvanovaN,PavlovM,XuB,SuZG,JansonJC.ChromatographicMethodsfortheIsolationof,andRefoldingofProteinsfromEscherichiacoliInclusionBodies,.ProteinExprPurif.,25(1):174-9,2002Zhenyu,Gu.Marianne,N.Bingze,Xu.Jan-Christer,Janson.Zhiguo,Su.“NewmethodsofusinggelfiltrationtoisolateandrenaturatethescFvinclusionbody”,ProteinExpressions&Purifications25(1):174-9,2002ProteinExpressions&PurificationsTOP5download2002MingLi,Zhi-GuoSu,andJan-ChristerJanson.Invitroproteinrefoldingbychromatographicprocedures,ProteinExpressionandPurification,33(1):1-10,200410010αRtotal=R1•R2•R2•••Rn•ktoteAA−•=ProcessIntegration)pH1mg/ml1mg/ml\pH/ampholytespH—1/2EPO3¾¾¾Startingwork-hemoglobinpurificationwithexpandedbedadsorption(EBA)Frombovine,porcineandhumanRBCProceduresofEBAPackedBedStablizedExp.BedFeedLoading&WashingElutionRegenerationProcesscomparisonExpandedbedadsorption(EBA)Membranefiltration+packedbedadsorption(MF+PBA)Numberofsteps12Timeh24Purityobtained9999Recovery8578EBAMF+PBA••••••(NH4)2SO4(%)05810121516PEG4000(%)10101010101010CellDisruptorCellDisruptorExtractorPEG&SaltsCellsuspensionCellsuspensionPEG&SaltsCentrifugeATPEafterCellDisruptionSimultaneousCellDisruption&ATPECentrifuge012345Disruptiontime(min)051015202530inwaterinPEG&saltsolutionProteinconcentrationinhomogenate(mg/ml)012345Disruptiontime(min)020406080100120ADHactivity(u/ml)InfluenceofdifferentdisruptionsolutionsonproteinreleaseandenzymaticactivityA:4mMK2HPO4/150mMNaCl(as100%),B:18%PEG600/17%(NH4)2SO4C:14%PEG1540/15%K2HPO4,D:10%PEG4000/9%(NH4)2SO4A:4mMABCD04080120160200Relativevalues(%)ProteinconcentrationEnzymeactivity0510152025ConcentrationofPEG600(%)020406080100120ADHactivityinhomogenate(u/ml)102030405060708090100110120130140150NH2COOHValArgSerSerSerArgThrProSerAspLysProValAlaHisValValValAsnGlnProAlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeuLeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSerGluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGlyGlnCysProSerThrHisValLeuLeuThrHisThrIleSerArgIleAlaValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSerProCysGlnArgGluThrProGluGlyAlaGluAlaLysProTrpTyrGluProIleTyrLeuGlyGlyValPheGlnLeuGluLysGlyAspArgLeuSerAlaGluIleAsnArgProAspTyrLeuAspPheAlaGluSerGlyGlnValPheTyrGlyIleIleLeuAlaαSSDisrupterCellsuspension20%Cellpaste,buffer,1%PEG1000CentrifugeCelldebrisIEX:DEAE-SepharoseFFGF:Superdex75IEXcolumnGFcolumnImpuritiesFurtherprocessing•••TimasheffPreferentialhydration¾¾¾()()32'2,,23131υρφφρµµooooTgg−−=⎟⎟⎠⎞⎜⎜⎝⎛∂∂[e-mail]