PurificationofKHCMotorDomainProtein IfyouusemethodsfromtheKinesinsite,weaskthatyoucitetheKinesinHomePageandauthors,ortheappropriatesourcepublicationinyourwork.Materials
SpecialEquipmentInducedcells(2-5gpelletofpET/KHCinBL21(DE3)pLysShostcells) HEMbuffer= 10mMHEPESpH7.21mMDTT1mMMgCL21mMEGTA HEM+25mMNaCl HEM+50mMNaCl HEM+100mMNaCl HEM+200mMNaCl 200mMPMSFinEtOH ProteaseInhibitorCocktail(200X)= 1microgram/mLPepstatinA1microgram/mLLeupeptin2microgram/mLAprotinin2microgram/mLTAME 1MDTT 1MMgCL2 10mg/mLDNAseI SPSepharosecolumn(2cmdiametercolumncontaining2-3mLresin) QSepharosecolumn(2cmdiametercolumncontaining2-3mLresin) Centricon30spinconcentrator(Amicon) Superose12FPLCcolumn(Pharmaciaorcomparable)
ProcedureBeckmanTLXultracentrifugeandTLArotor,orcomparable PharmaciaorcomparableFPLCsystem
Notes1. InducecellsandharvestasdescribedinBacterialExpressionofMotors.WashinducedcellswithHEM+25mMNaCl+0.5mMPMSFandfreezein30mLOakRidgecentrifugetubesat-80°Cuntiluse. 2. ResUSPendfrozencellsoniceat1-1.25mL/gofHEM+25mMNaCl.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+25mMNaCl.Washcolumnextensively(~8mL)withHEM+25mMNaCl. 8. ElutecolumnwithHEM+100mMNaCl(5x1mLfractions),thenwithHEM+200mMNaCl(8x1mLfractions).Thebulkoftheproteinstartstoelutewith100mMNaClinthe2ndfraction.Add5microlitersof200mMPMSFtoeachpeakfraction. 9. Poolpeakfractions,dilute1:1withHEMandloadontoaQSepharosecolumnequilibratedwithHEM+50mMNaCl.WashcolumnextensivelywithHEM+50mMNaCl. 10. ElutecolumnwithHEM+100mMNaCl(3x1mLfractions),thenwithHEM+200mMNaCl(8x1mLfractions).Theproteinpeakeluteswith200mMNaClinthe2ndto5thfractions. 11. Iffurtherpurificationisnecessary,reducevolumeusingaCentricon30spincolumn,thenadd1volumeHEMtoreduceNaClconcentrationto100mM. 12. ApplytoSuperose12FPLCcolumnequilibratedinHEM+100mMNaCl.Collect0.5mLfractions.Poolpeakfractions(fr26-28).FreezeinliquidN2andstoreat-80°C.
HSong&SAEndow2/991. KHCandotherkinesinmotorproteinsbindtoSP-Sepharose,whilemostbacterialproteinsflowthrough.ChromatographyonSP-Sepharoseisthereforeanimportantpurificationstepandcanyieldfractionsof90-95%homogeneity. 2. TheSPSepharoseandQSepharosecolumnscanbeprewashedwith5MNaClandequilibratedinHEM+25mMNaClorHEM+50mMNaCl.Aftereachuse,washwith10-20mLHEM+5MNaClfollowedby10-20mLofHEM+25mMNaClorHEM+50mMNaCl. 3. TheSPSepharoseandQSepharosecolumnfractionscanberapidlyassayedbyusingtheBio-Radproteinconcentrationreagenttoidentifythepeakfractions.Add5microlitersofeachfractionto395microlitersHEM+100microlitersof1:4dilutedBio-RadreagentandreadOD595relativetoacontrolwithnoaddedprotein. 4. ThepeakfractionfromSPSepharoseis~3-5mg/mLKHCmotordomainproteinstartingfrom2-5gcells.