PurificationofKar3MotorDomainProtein

Materials

Inducedcells(2-5gpelletofpET/Kar3inBL31(DE3)pLysShostcells)(Seenote#1)

HEMbuffer=

10mMHEPESpH7.21mMMgCL21mMDTT1mMEGTA

HEM+80mMNaCl

HEM+100mMNaCl

HEM+200mMNaCl

200mMPMSFinEtOH

ProteaseInhibitorCocktail(200X)=

1microgram/mLPepstatin1microgram/mLLeupeptin2micrograms/mLAprotinin2micrograms/mLTAME

1MDTT

1MMgCl2

10micrograms/mLDNAseI

SPSepharosecolumn(2-3mLbedvolume)

Centricon30spinconcentrator(Amicon)

MonoScolumn(Pharmaciaorcomparable)

Superose12FPLCcolumn(Pharmaciaorcomparable)

SpecialEquipment

BeckmanTLXultracentrifugeandTLArotor,orcomparable

PharmaciaorcomparableFPLCsystem

Procedure

1.	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(6x1mLfractions).Thebulkoftheproteineluteswith200mMNaClinthe2ndto4thfractions(fr200-2to200-4)andcanbe~90-95%pure.Add5microlitersof200mMPMSFtoeachpeakfraction.
9.	Poolpeakfractions,thenadd2volumesHEMtoreduceNaClconcentrationto<70mM.
10.	ApplytoMonoScolumnequilibratedinHEM.ElutecolumnwithalineargrADIentof0-500mMNaCl.TheKar3proteinelutesasamajorpeakat200mMNaClandaminorpeakat230mMNaCl.BothpeaksconsistofhighlypurifiedKar3motordomainproteinbySDS-PAGE.
11.	MinorcontaminantspresentinthemajorpeakofKar3fromtheMonoScolumncanberemovedbychromatographyonaSuperose12FPLCcolumnequilibratedinHEM+100mMNaCl.ReducevolumeusingaCentricon30spincolumnandadjustsaltconcentrationto100mMNaClbeforeloadingontothecolumn.Collect0.5mLfractions.Poolpeakfractions(fr26-28).FreezeinliquidN2andstoreat-80°C.

Notes

1.	TheKar3motordomaincomprises347aminoacidsandcorrespondstoresidues383-729ofS.cerevisiaeKar3.ThecodingregionoftheKar3motordomaincontains10ArgAGAcodons.Forexpressionofprotein,Kar3wasclonedintopMW172(Wayetal.1990)andco-transformedwithpACYC184/argU+(Spanjaardetal.1990)intocompetenthostcells.argU+istheE.coligenefortRNAArgAGA,whichislimitinginE.coli.ThecellsaregrowninM9ZBmediumcontaining5micrograms/mLoftetracyclineinadditiontoampicilinandCAP.
2.	Kar3andotherkinesinmotorproteinsbindtoSP-Sepharose,whilemostbacterialproteinsflowthrough.ChromatographyonSP-Sepharoseisthereforeanimportantpurificationstepandcanyieldfractionsof90-95%homogeneity.
3.	TheSPSepharosecolumncanbeprewashedwith5MNaClandequilibratedinHEM+80mMNaCl.Aftereachuse,washwith10-20mLHEM+5MNaClfollowedby10-20mLofHEM+80mMNaCl.
4.	TheSPSepharosecolumnfractionscanberapidlyassayedbyusingtheBio-Radproteinconcentrationreagenttoidentifythepeakfractions.Add5microlitersofeachfractionto395microlitersHEM+100microlitersof1:4dilutedBio-RadreagentandreadOD595relativetoacontrolwithnoaddedprotein.

HSong&SAEndow1997

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