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ISSN100727626CN1123870öQChin.J.Biochem.Mol.Biol.2000,Vol.16,No.1,1722161200023(940586)33Tel:(0759)2388501,Fax:(0759)2284104,E2mail:gdmcpres@gdmc.edu.cngdmcsh@gdmc.edu.cn,,196410,,,:1999203223,:1999207214CK2A333(,524023)(,510089)CK2AcDNA,BL21(DE3),IPTG,30%,LDE252P112Sepharose,309mg611mgLSDS2PAGE42kDLWestern2blot:CK2ALCK2ABLCK21CK2ALCK2A,,Q55,Q812ProkaryoticExpression,PurificationandCharacterizationofRecombinantHumanProteinKinaseCK2ASubunitLIUXin2guang,LIANGNian2ci(InstituteofMedicalBiochemistry,GuangdongMedicalCollege,Zhanjiang524023)MAJian2quan(DepartmentofBiochemistry,SunYat2senUniversityofMedicalSciences,Guangzhou510089)AbstractTherecombinantplasmidcontaininghumanproteinkinaseCK2AsubunitcDNAcon2structedsuccessfullywastransformedintoEscherichiacoliBL21(DE3)andexpressedsignificant2lyandspecificallyinducedbyIPTG.Therecombinantproteinwascomposedofapproximately30%ofthetotalbacterialproteins,however,mostofrecombinantwhichwereinsoluble.There2combinanthumanCK2AsubunitsequentiallywaspurifiedbyDE252,P11phosphocelluloseandHeparin2Sepharosechromatography.From309mgsolubleproteins,theyieldoftheCK2Aproteinwas6.1mg.SDS2PAGEanalysisofthepurifiedrecombinantproteinshowedonlyonebandwithmolecularmassof42kDinagreementwithnativehumanCK2Asubunit.Westernblotresultcon2firmedthattherecombinantproductcouldspecificallyreactwithantibodyagainsthumanCK2Asubunit.TheCK2AandBsubunitsweremixedatthesamemolarratio.TheproducedCK2holoenzymedisplayedthefullactivity.ThecharacterizationsandfunctionsofreconstitutedCK2holoenzymewereconsistentwiththoseofthegivennativeCK2.TheseresultsdemonstratedthattherecombinantproteinwashumanproteinkinaseCK2Asubunit.KeywordsHumanproteinkinaseCK2Asubunit,Prokaryoticexpression,Proteinpurification©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.CK2ö,(AA)(B)[1,2]LcDNACK2AAB391,350215[3]LCK2A,AB4244kD,38kD26kD[1,3]LCK2ATPGTP11954,[1,2]LCK2[3].[4,5],CK2A,,LNiefind[6]CK2A,CK2,L,LCK2,,,,CK2LCK2A,:111111111BL21(DE3),pT7-7Tabor[7]L11112(IPTG),5,62212B2D2(DRB)Cal2biochem,CK2A(McAb)BoehringerMannheim,CK2BMcAbIssingerL(NC)Sigma,DE252,P11P81Whatman,5mlEcono2PacBio2RadLABC(AP)L[C232P]ATP[C232P]GTPL11211211CK2AcDNApTCKApT727,[8,9]L11212DNApTCKABL21(DE3),A595015,IPTG(1mmolöL),4h,,-20L11213CK2A3000ml(6g)140mlA(20mmolöLTris2HCl,pH7.5,1mmolöLEDTA,0.5mmolöLEGTA,7mmolöLB2,0105molöLNaCl,0.5mgöL,017mgöLA),DNaseÉDNAL30000g,410min,(P2É),(S2É)150000g,43h,(P2Ê),(S2Ê)L11214CK2A4L0105molöLNaClADE252(115cm24cm),S2ÊLBio2RadEcono,2175ml0105016molöLNaClA,CK2,(01150130molöLNaCl),0125molöLNaClAP11(115cm13cm),2100ml0125112molöLNaClA,(016018molöLNaCl),NaCl015molöL,015molöLNaClAEcono2Pac(5ml),225ml015110molöLNaClA,1ml,(016molöLNaCl),L11215CK2[10,11]L35Ll,50LmolöLATP,[C232P]ATP[C232P]GTP0.5LCi(5000Ciömmol),2göLL15Ll,3010min30LlP81,85mmolöL,L1pmol[C232P]ATP[C232P]GTP32P1(U)L11216SDS2PAGELaemmli,0175mm12%L8116©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.11217SDS2PAGE,LCK2AL11218WesternblotCK2AMcAbABC(AP),NCBSA,CK2AMcAb()()Fig.1ExpressionofrecombinanthumanproteinkinaseCK2AsubunitinthetransformedbacteriaA:CrudeextractofbacteriatransformedbypT727;B:CrudeextractofuninducedbacteriatransformedbypTCKA;C:CrudeextractofIPTG2inducedbacteriatransformedbypTCKA;D:P2ÉfractionofC;E:S2ÉfractionofC;F:P2ÊfractionofC;G:S2ÊfractionofC;H:Proteinmolecularweightstandard(AP),NBTBCIP,L2211CK2ACK2ApTCKABL21(DE3),IPTG42kD(Fig.1,C),CK2A30%(Fig.2),IPTGCK2A(Fig.1,B),pT727CK2A(Fig.1,A),LFig.1CK2ALFig.2ThescanninggraphofSDS2PAGEofcrudeextractofIPTG2inducedbacteriatransformedbypTCKA212CK2ATable1.Table1PurificationofrecombinanthumanproteinkinaseCK2AsubunitPurificationstepVolumeömlTotalproteinömgTotalactivityöUSpecificactivityöUmg-1S2Ê130309783215DE252140189124736610P115522824637418Heparin2Sepharose36112634431183L(6g)309mg,3611mgLS2ÊCK2,,L2133,SDS2PAGE(Fig.3):42kD(B),[1,3],L214Westernblot(Fig.3):CK2AMcAb,NC42kD(A),NCCK2B911:CK2A©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Fig.3SDS2PAGEofpurifiedrecombinanthumanproteinkinaseCK2AsubunitanditsWesternblotA:WesternblotforpurifiedrecombinanthumanCK2Asubunit;B:PurifiedrecombinanthumanCK2Asubunit;C:ProteinmolecularweightstandardMcAb()1CK2BMcAb,CK2AL21521511CK2AKm(2göL),ATPGTP(1215,25,50,100LmolöL),ATPGTP,CK2A,CK2A28pmol,CK2,ATPGTP,Hanes2Woolf(Fig.4)KmLCK2AATPGTPKm117LmolöL3114LmolöLL21512CK2Fig.4Hanes2WoolfplotfortheKmofCK2AsubunitTheassaywascarriedoutwith28pmolpurifiedCK2A,us2ingcasein(2göL)asphosphateacceptorandATP()orGTP()asphosphatedonorCK2(AA)(B)LCK2ACK2B(),,(Table2)CK2BCK2,ABCK2CKA4,CK2BCK2LCK2BCK2A,ABLTable2ActivationoftheCK2AsubunitbyCK2BsubunitRatioAöB(molömol)CK2activityö10-3U(xqs)%ofCK2Aactivity10326.533.210010.2682.4109.02091)10.61093.293.53351)11.01282.976.63931)11.41321.1112.24051),2)11.81353.646.44151),2)ActivityassayoftheCK2AsubunitwasperformedusingaconstantamountofCK2Asubunit(28pmol)andincreasingamountsofBsubunit(5.6pmol50.4pmol).n=3,1)P0.01(comparisonwithAB=10group),2)P0.05(comparisonwithAB=11.0group)21513CK2Table3.,CK2CK2Table3CharacterizationofrecombinanthumanCK2holoenzymeGroup