PurificationofNcdMotorDomainProtein

Materials

Inducedcells(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)

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(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.

Notes

1.	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.

HSong&SAEndow2/99

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