PurificationofCD2

1)Cellsarepelleted(~3-4000gfor10min.)andresUSPendedinpre-chilled50mMmalonate(pH5.2-5.3)with1mMEDTA(~40mlofbufferperlitreofculture).

2)LysethecellswithFrenchpress.

3)Pelletthecelldebris(~20000gfor30min.).Carefullytransferthesupernatanttoanewtube.CheckpH(5.4~5.5).

4)EquilibratetheSP-sepharosecolumnwiththemalonatebuffer(~1-1.5houratflowrateof2ml/min).LoadthecrudecelllysateontoSPcolumn,washandtheneluteusingasaltconcentrationgrADIentontheFPLC(programme5).

5)AnalysetheeluatewithSDS-PAGEandpoolthedesiredfractions.

6)Equilibratethegel-filtrationcolumnwith20mMphosphate(pH5.4)for2hoursat1-1.5ml/min.Loadthepooledfractionsontothegel-filtrationcolumn(load~5mlperrun).Thisshoulddesaltandbuffer-exchangetheCD2proteinaswellasremovingthelargercontaminants.

7)Analysethefractionscollected.Ifnecessary,loadthepooledfractionsontotheS-10cationexchangecolumnandeluteusingastepwisepHelution.

9)ConcentrateproteinusingCentriprep(~1000g)toafinalconcentrationof1-2mM.Theproteinhasatendencytoprecipitateathighconcentration(especiallybetweenpH5to7),thereforetakecaretoconcentrateslowly.Expectproteinyieldof~20-40mg/Lifthereislittleprecipitation.TheproteinthusobtainedispureenoughforNMRstudiesbutproteaseinhibitors(PMSF+EDTA)needtobeadded(Itispreferablethatallbuffersusedinthepurificationshouldcontain0.1mMPMSFand0.5mMEDTA).

Note:i)CD2shouldbepurifiedasquicklyaspossIBLetoavoidtheproblemofproteolysis.Donotstoredsampleinfridgeformorethanadaybetweenpurificationsteps,storedat-20°Cinstead.ii)Avoidasmanystepsaspossiblewhenpurifyingprotein.Proteinsarelostduringeachpurificationstep,thereforefewerstepsminimiselostofproteins.iii)Gelfiltration(sizeexclusion)isoftenusedasthefinalstepinproteinpurificationbuthereitisusedtodesaltandbufferexchangetheprotein(thereforenoneedfordialysis)aswellasremovingthecontaminatingproteinsoflargersizes.IftheCD2appearedpurified,nofurtherstepsarenecessary,otherwisepurifytheproteinfurtherusingamono-Scationexchangecolumn(eluteatpH6.5,7.5and8.5-CD2shouldeluteatpH7.5)orothercolumnssuchashydrophobicinteractioncolumn.iv)ForSPandMono-Scolumnsaftertheelutionoftheproteiniscompleted,washthecolumnwith1-2MNaClsolutiontoremoveanyboundproteins.Washagainwithlowsaltbufferandthenstorethecolumnin20%ethanol.v)TheSPcolomninfirststepinthepurificationmaybereplacedwithS-sepharose.Thisgiveasharperpeakandmayneedonlyafurthergel-filtrationstep.vi)TheelutionoftheproteinismonitoredwiththeODat280nmwhichistheabsorptionmaximumoftryptophan(themaincomponent)andtyrosine(whichabsorblessstronglythantrp).vii)ThepHofthecrudecelllysateiskeptat5.3toensurethattheproteinwillbindwelltotheSPcolumnbutnotsolowastoprecipitatemostoftheE.coliproteinsaswellasCD2.MostE.coliproteinshaveisoelectricpoint<5thereforetendtoprecipitateoutatlowpH-thiswouldbeusefulforremovingmostofunwantedbacterialproteinbutnotifyourproteinprecipitateoutaswell.Itis,however,usefultonotethatresultsindicatethatmaximaldimerformationofCD2(andwhichappearedtobeassociatedwiththeprecipitationofCD2)occursat~pH6(actuallyprobablypH5.7)whileatpH<5,monomerpredominates.ItmaythereforebepossibletostartalowerpH(say~4-4.5),buttherewillbeconsiderableprecipitationofbacterialproteinswhichmaycarrytheriskofco-precipitatingCD2aswell.viii)WithprecipitationthemainproblemsinCD2purification,itmaybeworthconsideringusingtheHis-tagconstructwhichispurifiedatahigherpHwherelessprecipitationisobserved.Allthewild-typeandmutantCD2areconstructedinboththenativeandhis-tagversions.Thisisespeciallyusefulforsomemutantswhichprecipitateheavilyinacidiccondition(e.g.K43A,L38A,Y81A?)usingthenormalmethodofpurification.ThereforeusetheHis-tagpurificationmethodforthehis-taggedversionofCD2(notethatthereshouldn"tbeanyEDTAinthebufferinthisfirststepofpurification).Add~1-5mgofcarboxypeptidaseaftertheHis-tagpurificationstepandincubateifyouwishtoremove(oneortwohistineresidues,however,willremain).Thisisfollowedbyagel-filtrationstep.IfyouarepurifyingthemutantproteinsthatmayprecipitateheavilyinacidicpH,thenitisadvisablenottousetheS-10columnnext.Thefirst2stepsarenormallysufficienttogetareasonablypureproteinifthehis-tagstepisdonecarefully.However,iftheproteinsamplestilllookimpureonthegel,loadtheHis-taggedprotein(thehis-tagshouldnotbecleavedforthisandmaynotworkforsomeofthemutants)ontotheS-10columnatpH6.5(theremaynotbesomuchprecipitationatthispH),thenwashwithpH7.5,andeluteatpH8.5.