趋化因子新变异体MPIF21β重组蛋白的纯化和活性分析

[]100028861(2002)0420311204[]2002203212;[]2002204227[](1955-),,,,,Tel:[phone];E2mail:[e-mail]211,1,2,2,2,2(1.,100083;2.,100083)[]MPIF21,pKPL3a2MPIF21MPIF21,15%,,,SepharoseCL26B,CMSepharoseFastFlowSephacrylS2200SDS2PAGE15000u,99.0%,6.6,9.07%MPIF21,U937,MPIF21,MPIF21[];MPIF21;MPIF21;[]R392[]APurificationandfunctionalcharacterizationofrecombinantMPIF21,anovelisoformofhumanchemokineMPIF21FANHui1,MOXiao2ning1,CAOLi2,SONGQuan2sheng2,HUANGDa2wu2,MADa2long2(1.BeijngMedicalUniversityUnionIncorporation,Beijing100083,China;2.PekingUniversityCenterforHumanDiseaseGenomics,Beijing100083,China)[Abstract]ObjectiveInordertostudythestructureandfunctionofMPIF21,anewisoformofhumanMPIF21,recombinantproteinwasexpressedinE.colianditspurifyingprocedurewasdetermined.MethodsItwasfoundthattherecombinantproteinpresentedasinclu2sionbodyandhadanexpressinglevelupto15%.ForthepurificationoftheMPIF21,theinclusionbodywaswashed,denaturedandrena2tured,andthenthreestepsofchromatography(heparinaffinitychromatography,CMionexchangeandSephacrylS2200sizeexclusion)werecarriedouttoobtainpurifiedprotein.ResultsSDS2PAGEanalysisshowedasinglebandatmolecularweight15000u.Thepurityoftargetproteinwasupto99.0%,thetotalrecoveryrateis9.07%.BioassayinvitroshowedMPIF21couldinhibitmouseLPP2CFCandattractU937cellswithdose2dependentmanner.ConclusionsAsimplymethodofpurifiedrecombinantMPIF21proteinisestablishedanditsbiologicalac2tivityhasbeenfound.[Keywords]chemokine;MPIF21;MPIF21;recombinantprotein21(myeloidprogenitorin2hibitoryfactor1,MPIF21),,[1]MPIF21,cDNAMPIF21,MPIF2151(17),,MPIF21MPIF21,()MPIF21,,11.1MPIF21pKPL3a2MPIF1[2],Pop2136C600(CI857,),,Raiband1.2GradifracSepharoseCL26B,CMSepharoseFastFlow,11318420027IMMUNOLOGICALJOURNALVol18No4July2002©1995-2003TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.SephacrylS2200Phamarcia1.3MPIF21Pop2136,,30A6000.6423.5h10000g10min;[3]8mol/L(20mmol/L2ME),603h30000g12minSepharoseCL26B,0.5mol/LNaCl110pH7.225mmol/LPBCMSepharoseFastFlow0.28mol/LNaClSephacrylS2200,pH7.2PBSMPIF211.4MPIF21LowrrySDS2PAGE3%,15%8852501.5MPIF211.5.1MPIF21:6C57,,,MEM,,MEM1,10%MEM(15ng/mLIL21,7.5ng/mLIL23,150ng/mLSCF)5103/mL35mm,1mL,3,37CO215(501000)1.5.2MPIF21U937:[4]U9372106/mL,,MPIF21,3372hGiemsa,22.1MPIF21MPIF21,,SepharoseCL26B,0.5mol/LNaCl(1)CMSepharoseFastFlow0.28mol/LNaCl(2)SephacrylS2200(3)SDS2PAGE,GeneSYSGENE99%,15000u1sepharoseCL26BMPIF21Fig1HeparinsepharoseCL26BchromatographicprofileofMPIF212CMsepharoseFFMPIF21Fig2CMsepharoseFFionexchangechromatographicprofileofMPIF213SephacrylS2200GelMPIF21Fig3SephacrylS2200GelfiltrationchromatographicprofileofMPIF21MPIF2115%6.6,9.07%14MPIF212131820027©1995-2003TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,,MPIF2195%0.2mpH8.0pH4.0MPIF21(5)1MPIF21Tab1SummaryofpurificationofMPIF21steptotalprotein/mgspecificprotein/(%)purificationfoldengineeredE.coli15indusionbodies67.4433.52.23heparinsepharose22.1771.84.78CL26BCMsepharoseFF6.4497.36.48sephacrylS22006.1299.06.601E.coli2136/MPIF21control;2E.coli2136/MPIF21induced;3MPIF21indisionbodies;4HeparinSepharoseCL26BpurifiedMPIF21;5CMSepharoseFFpurifiedMPIF21;6SephacrylS2200purifiedMPIF21;7proteinmolecu2larmarker4MPIF21SDS2PAGEFig4SDS2PAGEanalysisofpurifiedMPIF211denaturingsolutionwithoutfiltering;2filtereddenaturingsolution;3renaturingsolutionwithoutfiltering;4filteredre2naturingsolution(pH4.0);5filteredrenaturingsolution(pH5.5);6filteredrenaturingsolution(pH7.2);7filteredrenaturingsolution(pH8.0);8proteinmolecularmarker5MPIF21SDS2PAGEFig5SDS2PAGEanalysisofrenaturingMPIF212.2MPIF212.2.1:MPIF21,,,22MPIF21Tab2InhibitionofMPIF21onmousemeyloidprogenetorcloneformationsamplecloneformationinhibitionrate/(%)MPIF21(250ng/L)20664.29MPIF21(50ng/L)25355.36MPIF21(10ng/L)41226.79PBS(pH7.2)56502.2.2MPIF21U937:MPIF21U937,66MPIF21U937Fig6ChemotaxiseffectofMPIF21onU937cells3,,,4:1CXC,2;2CC,2;3C424;CX3C,123[5]CC,CXC,C,253134,1MPIF21©1995-2003TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,MIP21,IL28,MPIF21,,[6,7]MPIF21,MPIF21MPIF21,MPIF21,MPIF21MPIF21MPIF21,MPIF21,,MPIF21,,MPIF21,,MPIF21,MPIF21,U937,MPIF21MPIF21,,MPIF21,,MPIF21[][1]PatelVP,KreiderBL,LiY,etal.MolecularandfunctionalcharacterizationoftwonovelhumanC2Cchemokinesasinhi2bitorsoftwodistinctclassesofmyeloidprogenitors[J].JExpMed,1997,185(7):1163-1172.[2],,.[J].,1990,22(2):81-83.[3],,,.rhG2CSF2Ala217[J].,2000,32(4):337-339.[4]HanW,LouY,TangJ,etal.Molecularcloningandcha2racterizationofchemokine2likefactor(CKLF1),anovelhu2mancytokinewithuniquestructureandpotentialchemotaticactivity[J].BiochemJ,2001,357(1):127-135.[5],.CHAK:[J].,1999,15(4):282-284.[6]MarshallE,WoolfordLB,LordBI.ContinuousinfusionofmacrophageinflammatoryproteinMIP21enhancesleucocyterecoveryandhaemopoieticprogenitorcellmobilizationaftercyclophosphamide[J].BrJCancer,1997,75(12):1715-1720.[7]BernsteinSH,EavesCJ,HerzigR,etal.ArandomizedphasestudyofBB210010:avariantofhumanmacrophageinflammatoryprotein21alphaforpatientsreceivinghigh2doseetoposideandcyclophosphamideformalignantlymphomaandbreastcancer[J].BrJHaematol,1997,99(4):888-895.()(3