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1、PurificationofinclusionbodiesandrefoldingofproteinsBasicStrongLabprotocol,basedonarecipeconcoctedbyPingweiLi,(cite,ifused:Steinle,A.,Li,P.,Morris,D.L.,Groh,V.,Lanier,L.L.,Strong,R.K.&Spies,T.(2001)‘InteractionsofhumanNKG2DwithitsligandsMICA,MICB,andhom
2、ologsofthemouseRAE-1proteinfamily’Immunogenetics53,pp.279-87)aderivativeofarecipeoriginallydevelopedintheJoneslab(O'CallaghanCA,TormoJ,WillcoxBE,BlundellCD,JakobsenBK,StuartDI,etal.(1998)'Production,crystallization,andpreliminaryx-rayanalysisofthehuman
3、MHCclassIbmoleculeHLA-E'ProteinScience7,pp.1264-1266)++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++1.Preparationofinclusionbodies:a.Harvestbacteriaafterinductionforabout4hours.(WegenerallyinduceexpressionatODofabout1.0forinclu
4、sionbodypreps,thismayimprovetheyieldalot).Troubleshootingforgoodproteinexpressioncanincludethefollowing:--Expressionatroomtemporat16degreesratherthan37.--Expressionfromafreshlytransfectedstrainratherthanfrozenglycerolstock.b.Lysebacteriabysonicationint
5、hefollowingbuffer:*50mMTris-HCl,pH8.0100mMNaCl5mMEDTA0.1%NaN30.5%Triton-X100(canusea25%stock)add0.1mMPMSFand1mMDTTimmediatelybeforeuseGenerallywelysecellsfrom4litersofprepin250mloflysingbufferandwashwith250mlofthesamebufferoverthenextthreesteps.50-mLal
6、iquotsworkwellforsonication.(*=Youcanalsouseamicrofluidizeratthisstep.)c.Aftersonication,add10mMMgSO4(use2.0Mstock)tochelatetheEDTA,thenaddDNase(about0.01mg/ml)andlysozymetoabout0.1mg/mltothelysateandincubateatRTfor20min.d.Centrifugetocollectinclusionb
7、odies(forexample,6000rpmfor15minutes).Crushthepelletwithaspatula,thenresuspenditcompletelybysonicationinthelysingbuffer.AnotherportionofDNaseandlysozymecanbeaddedatthispointtoimprovethepurityofthepellet.e.RepeatstepdtwomoretimeswithoutaddingDNaseandlys
8、ozyme.f.WashtheinclusionbodyagainwithlysingbufferwithoutTriton-X100.Resuspendthispelletbysonicationaswell.Bufferforfinalwash(noTriton-X):50mMTris-HCl,pH8.0100mMNaCl5mMEDTA0.1%NaN3g.Collectthefinalinclusionbodypelletbycentrifugationin50-
