Introduction

Highlevelsofexpressionofrecombinantproteinsinabacterialsystemcanleadtotheformationofinsolubleaggregates,usuallyknownasinclusionbodies(IB).6MGuanidine-HCl(GuHCl),8MUreaorotherstrongdenaturantscanbeusedtocompletelysolubilizedIB.SinceunderdenaturatingconditionstheHistagiscompletelyexposed,itwillfacilitatethebindingtoNicolumns.Formostbiochemicalstudies,proteinhavetoberenaturedandrefolded,andthiscanbedoneinthecolumnitselfbeforeelutionorafterelution.

AliquotofCellPelletafterInduction

Theideaistoaliquotcellsafterinduction,andkeepat-80ºCenoughcellpelletsamplesforoptimizationofsmallscalepurificationprocedureandfurtherscale-up.Onceyousetupthebestpurificationconditionsatlowscale,youcanscale-uptheprocedure.

EquilibrationofNi-NTAagarose

Place50ulbeads(100ulsuspension)ofNi-NTAagarosebeadsin1.5mlplastictube.

Washwith2x1.5mlH₂Oand2x1.5mlequilibrationbuffer(washing:mix,spin3min3500rpm,dischargesupernatant).

Buffers

Lysisbuffer:50mMNa₂HPO₄pH8.0,0.3MNaCl,1mMPMSF(orproteaseinhibitorcocktailforbacterialcells#P-8849fromSigma)andstrongdenaturantas6MGuanidine-HCl(GuHCl)or6to8MUreaOptionaladditivestothelysisbuffera)1mMPMSForproteaseinhibitorcocktail1:200(cocktailforbacterialcells#P-8849fromSigma)b)Dnase100U/mlor25-50ug/ml(SIGMADN-25).Incubate10min4°Cinthepresenceof10mMMgCl₂

Equilibrationbuffer:6to8MUrea,50mMNa₂HPO₄pH8.0,0.5MNaCl

Washingbuffer:6to8MUrea,50mMNa₂HPO₄pH8.0,0.5MNaCl

Elutionbuffer:6to8MUrea,20mMTrispH7.5,100mMNaCl,andappropriateimidazoleconcentrations.

LimitationsDonotexposedNimatricestoreducingagentsasDTTorDTE(youcanuseßMEupto20mM);chelatingagentsasEDTAandEGTA;NH₄⁺buffersandaminoacidsasArg,Glu,GlyorHis.

Procedure

1Resuspendpelletof10mlbacterialculture(or100mlbacterialcultureforverylowexpressionlevel)in1mllysisbuffer

2Preparelysisbuffercontainingurea6to8MorGuanidine-HCl6M(try8MofUreafirst,andifproteinissolubletiterdowninthenextexperimentstillureaconcentrationtillminimalureaisrequiredforproteinsolubilization)

3Sonicateonice3x20seconds(dependsofthesonicator)

4Spin15minmaxspeed4°C

5Transfersupernatantintocleantube:crudeextract(keep40µlforPAGE-SDS)

6Equilibrate50µlNilbeadswithequilibrationbuffer(seeEquilibrationofNi-NTAagarose)

7Addthecrudeextracttothebeadsandincubate4°C/1h(swirl)

8Spin3min3500rpm.Dichargeunboundmaterial(keep40µlforPAGE-SDS)

9Wash3x1mlwithwashbufferwithappropriateureaconcentration.Washing:mix,spin3min3500rpm,dischargesupernatant(keep40µlforPAGE-SDS)

10Wash2x1mlwithwashbuffer+10mMimidazole(keep40µlforPAGE-SDS)

11Elutewith2x100µlelutionbuffer+100mMimidazole(keep40µlforPAGE-SDS)

12Finalelution:2x100µl250mMimidazole(keep40µlforPAGE-SDS)

13RunonPAGE-SDSgel5µlofcrudeextractandunboundmaterial,and13µlofthewashandelutionfractions.

Analysisofresults-Troubleshooting

IfproteindoesnotbindtotheNi-NTAresin,checktheintegrityoftheHistagbywesternblotusinganti-polyhistidineantibodies,ordoN-terminalsequencinginthecaseofN-terminaltags.Trytoworkat4°Callthetimeusingproteaseinhibitorsduringlysis.

IftargetproteinelutewithproteincontaminantstrythealternativeprotocolwithincreasingImidazolconcentration.OrreduceamountofNi-NTAresin.OrtryadditivesasßME,glycerol,detergentsormoreNaClinthewashing/elutionbuffers.

Ifelutedproteinseemtobedegradedtrytoworkat4°Callthetimeanduseproteaseinhibitorsduringlysis.

Iftheproteindoesnotelutefromthecolumn(proteinisattachedtothecolumn)usehigherImidazolconcentrations(upto1M).

Alternativeprotocoliftargetproteinisnotpureenough

Performparallelpurificationprocedureswhereyouinclude10,20,30,40or50mMimidazoleinthelysis,bindingandwashingbuffer.

Elutedirectlywith3x100ulelutionbuffer+250mMImidazol.

CheckelutedproteinsonPAGE-SDS.ExpectloweryieldsbuthigherpurificationbyincreasingtheImidazolconcentration

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