PurificationofKHCMotorDomainProtein
MaterialsInducedcells(2-5gpelletofpET/KHCinBL21(DE3)pLysShostcells) |
HEMbuffer= | 10mMHEPESpH7.21mMDTT1mMMgCL21mMEGTA |
ProteaseInhibitorCocktail(200X)= | 1microgram/mLPepstatinA1microgram/mLLeupeptin2microgram/mLAprotinin2microgram/mLTAME |
SPSepharosecolumn(2cmdiametercolumncontaining2-3mLresin) |
QSepharosecolumn(2cmdiametercolumncontaining2-3mLresin) |
Centricon30spinconcentrator(Amicon) |
Superose12FPLCcolumn(Pharmaciaorcomparable) |
SpecialEquipmentBeckmanTLXultracentrifugeandTLArotor,orcomparable |
PharmaciaorcomparableFPLCsystem |
Procedure1. | 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. |
Notes1. | 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. |
HSong&SAEndow2/99
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