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Purification of Ncd Motor Domain Protein
PurificationofNcdMotorDomainProtein IfyouusemethodsfromtheKinesinsite,weaskthatyoucitetheKinesinHomePageandauthors,ortheappropriatesourcepublicationinyourwork.Materials
SpecialEquipmentInducedcells(2-5gpelletofpET/NcdinBL21(DE3)pLysShostcells) HEMbuffer= 10mMHEPESpH7.21mMDTT1mMMgCL21mMEGTA HEM+80mMNaCl HEM+100mMNaCl HEM+200mMNaCl 200mMPMSFinEtOH ProteaseInhibitorCocktail(200X)= 1microgram/mLPepstatinA1microgram/mLLeupeptin2microgram/mLAprotinin2microgram/mLTAME 1MDTT 1MMgCL2 10mg/mLDNAseI SPSepharosecolumn(2cmdiametercolumncontaining2-3mLresin) Centricon30spinconcentrator(Amicon) Superose12FPLCcolumn(Pharmaciaorcomparable)
ProcedureBeckmanTLXultracentrifugeandTLArotor,orcomparable PharmaciaorcomparableFPLCsystem
Notes1. InducecellsandharvestasdescribedinBacterialExpressionofMotors.WashinducedcellswithHEM+80mMNaCl+0.5mMPMSFandfreezein30mLOakRidgecentrifugetubesat-80°Cuntiluse. 2. ResUSPendfrozencellsoniceat1-1.25mL/gofHEM+80mMNaCl.AddPMSFto1mMand1/200xvolumeproteaseinhibitors.Resuspendthecellsusingaglassrodandavoidfoaming. 3. Freeze/thawtoensurethecellsarelysedbyfreezingtubeinliquidN2for3-4min,thenswirlingina37°Cwaterbathuntiljustthawedandstillcold.Transfertoice. 4. AddDTTto0.5mM,anotheraliquotofPMSFandproteaseinhibitors,MgCl2to5mMandDNAseIto40micrograms/mL.Incubate15-20minonicetodigestDNA.AddanotheraliquotofPMSFandproteaseinhibitorshalfwaythroughtheincubation. 5. Centrifugeat18,000rpm(39,000xg)and4°Cfor20minintheSorvallSS-34rotor. 6. TransferclearyellowsupernatanttoBeckmanTLAultracentrifugetubes.Centrifugeat80,000rpm(270,000xg)and2°Cfor30minintheTLA100.3rotorinaBeckmanTLXultracentrifuge. 7. ApplyclearyellowsupernatanttoSP-Sepharosecolumnat4°CequilibratedwithHEM+80mMNaCl.Washcolumnextensively(~8mL)withHEM+80mMNaCl. 8. ElutecolumnwithHEM+100mMNaCl(3x1mLfractions),thenwithHEM+200mMNaCl(7x1mLfractions).Thebulkoftheproteineluteswith200mMNaClinthe2ndto4thfractions(fr200-2to200-4)andis~90-95%pure.Add5microlitersof200mMPMSFtoeachpeakfraction. 9. Poolpeakfractions,reducevolumeusingaCentricon30spincolumn,thenadd1volumeHEMtoreduceNaClconcentrationto100mM. 10. ApplytoSuperose12FPLCcolumnequilibratedinHEM+100mMNaCl.Collect0.5mLfractions.Poolpeakfractions(fr26-28).FreezeinliquidN2andstoreat-80°C.
HSong&SAEndow2/991. NcdandotherkinesinmotorproteinsbindtoSP-Sepharose,whilemostbacterialproteinsflowthrough.ChromatographyonSP-Sepharoseisthereforeanimportantpurificationstepandtypicallyyieldsfractionsof90-95%homogeneityfortheNcdmotordomainprotein(residues333-700). 2. TheSPSepharosecolumncanbeprewashedwith5MNaClandequilibratedinHEM+80mMNaCl.Aftereachuse,washwith10-20mLHEM+5MNaClfollowedby10-20mLofHEM+80mMNaCl. 3. TheSPSepharosecolumnfractionscanberapidlyassayedbyusingtheBio-Radproteinconcentrationreagenttoidentifythepeakfractions.Add5microlitersofeachfractionto395microlitersHEM+100microlitersof1:4dilutedBio-RadreagentandreadOD595relativetoacontrolwithnoaddedprotein. 4. ThepeakfractionfromSPSepharoseis~5-10mg/mLNcdmotordomainproteinstartingfrom2-5gcells.IftheproteinconcentrationoftheSPSepharoseunboundfractionis<20mg/mL,thecellswerenotlysedproperly. 5. TheNcdmotordomainproteintendstoaggregateandshouldbekeptin50-100mMNaClduringthepurification.Partiallypurifiedproteinundergoesdegradationwhenkeptonice>1-2hrs-thepurificationshouldbecarriedoutrapidlyandanySPSepharosecolumnfractionsthataresavedshouldbeflashfrozeninliquidN2andstoredat-80°C. 6. MissensemutantscontainingonlyasingleaminoacidchangecanbedramaticallyalteredintheirABIlitytobindtoSPSepharose.ItisthereforenecessarytocarryouttrialexperimentsforvariantormutantNcdproteinstodetermineiftheproteinbindstoSPSepharoseand,ifso,theNaClconcentrationatwhichtheproteinelutes.
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