GSTFusionProteinPrep

Italicsindicateoptionalstepsespeciallyusefulfortheanalysisofuntestedproteins.

GROWTHANDHARVESTINGOFBACTERIA

• Add100ulampicillinto100mlLB.Inoculateandgrowovernight.
• Inthemorning,addovernightbrothto900mlprewarmedLB.Grow1hourortoA600greaterthan0.5.Ampicillincanbeaddedtohelpmaintaintheplasmid.Makeglycerolstockfromovernightcultureifneeded!
• Add1ml0.1MIPTG(final0.1mM)tothe1Lculture.Grow3to5hoursortoA600greaterthan1.0.Priortoinduction,remove100ulcellsandadd100ulSDS-PAGEsamplebuffer.Boilbrieflyandload10ulforSDS-PAGE.SharperbandscanbeobtainedifyoupassthroughsyringeseveraltimestofragmentDNA.Collecthourlysamplestodetermineoptimalinductiontime.
• Pourcultureintotwo500mlcentrifugebottlesandpelletat5K10"4C.
• ResUSPendin10mlresuspensionbuffer.KeepbugsoniceasmuchaspossIBLefromhereonouttominimizeproteolysis.
• LysebacteriabypassingthroughtheFrenchPress2x@12,000psi.AddPMSFifdesiredjustbeforepressing.Save100ulofpressedlysate.Pellet10K10".Load2.5ulsupernatantforSDS-PAGE.Alsoresuspendpelletandload2.5ultocheckwhethertheproteinisinsoluble.
• Transferpressedlysatetoa15mlcentrifugetube.Pelletcellulardebrisat10K10"4C.Aliquotsupernatantandstoreat-70C.

BINDINGTOBEADSANDTHROMBINCLEAVAGE

• Incubatesolublelysatewith1/2volumeof50%GSTbeadsat4CrockingontheNutatorfor30-60minutes.50mlconicalFalcontubesworkwell.
• PourintoaBioRadColumnanddrainlysate.Save100ulofdepletedlysate.Load2.5ulforSDS-PAGE.
• Washbeadswith3columnvolumesresuspensionbufferand1volumethrombincleavagebuffer.
• Sealthebottomofthecolumn.Add1columnvolumethrombincleavagebufferplusthrombin.10ugpermgfusionproteinisagoodstartingpoint.Incubate30-60minutesatroomtemprockingontheNutator.Iftheproteinissubjecttodegradation,trya60minutecleavageat4C.
• DrainBioRadcolumns.Solublecleavedfusionproteinwillbeinthefiltrate.Addacolumnvolumeofbuffertorinseoutallcleavedprotein.Rinseandsavelabelledtubesforre-use.Saveabout20ulofbeads.Load1-10ulforSDS-PAGE.
• Optional:asecondthrombincleavagein1/2thepreviousvolume.
• InactivatethrombinwithAEBSForPMSF.ConcentrateontheCentriconorCellStirrerifneeded,orfreezeasis.Save100ulofunconcentrated,purifiedprotein.Load1-10ulforSDS-PAGE.Quantitatebycomparisonto2.5,1.0,and0.25mgBSAstandardsonthesamegel.

BINDINGTOBEADSANDTHROMBINCLEAVAGEAlternateMiniPrep

• Thawout1mlaliquotoffusionproteinlysate.Centrifugetoremoveprecipitate.
• Incubate1mllysatewith0.4ml50%GSTbeads.Incubateat4Cwithmixingfor1hour.
• Centrifuge5K2"in1.5mlEppendorftubes.Washthebeads4XwithPBST(0.01%bMEoptional)and2Xwiththrombincleavagebuffer.Stophereifproteinboundtobeadsisneededforbindingstudy.
• Resuspendthebeadsin150ulthrombincleavagebufferandadd1.4ugthrombin.Incubateatroomtemperaturewithmixingfor1hour.
• Recoverthesupernatantcontainingthefusionproteinbycentrifugation.Stopthereactionwith0.7ulPMSF.Incubateatroomtemperature15".Addanother0.7ulPMSFandincubateanother15".

MATERIALS

• PBST:PBS(Phosphate-BufferedSaline)+0.05%Tween20
• ResuspensionBuffer:PBST+2mMEDTA+0.1%bME
• ThrombinCleavageBuffer:AnybufferbetweenpH7-8.5withCa++isadequate.Try50mMTRISpH8.0+150mMNaCl+2.5mMCaCl2+0.1%bME
• 50%GlutathioneAgaroseBeads("GSTBeads")1.85gbeadsper50ml:Rehydratebeadsinlargevolumeofwaterforatleastanhour.WashwithseveralvolumesPBSTthroughWhatmanfilteronaBuchnerfunneltoremovemaltosestABIlizer.CollectgelandaddequalvolumeofPBST.Storeat4C.Donotfreeze-beadsarefragileandicecrystalswilldestroy.
• Ampicillin:1.0g/10mlsterileH2O,filter0.22mM,storeat-20Cin1mlaliquots.Use1:1000.
• IPTG:0.1Mstocksolution:0.238g/10mlsterileH2O,storeat-20Cin1mlaliquotsAlternate5xsolution:5g/42ml
• PMSF:0.3MstocksolutioninDMSO;use1:1000
• Kanamycin:0.25g/10ml,use1:1000onlyforcellswith[pREP-4]plasmid!

ORDERINGINFO

• Sigma:Glutathionebeads,cat#G-4510
• Sigma:Thrombin,3560NIHunits/mgprotein,cat#T-6759
• ICN:AEBSF:#193503

NOTES

• bMEshouldbefreshlyaddedwhencalledfor,asitisrapidlyoxidized.
• PMSFshouldbeaddedjustpriortolysisofbacteria,asitisunstableinaqueoussolutions.
• InsteadofPMSFtryless-toxicAEBSF.Stocksolution:0.2MinH2O,use1:100
• nSec1,astickyprotein,givesahigheryieldif0.5MNaClisaddedtotheresuspensionbufferand0.05%Tween20totheThrombinCleavageBuffer.Sincetheresuspensionbufferisalready150mMNaCljustadd0.35MNaCl.Ifyouintendtofreezethepressedlysate,leaveinthecelldebris.Centrifugeitoutpriortopurification.
• StirredCellConcentrator:SoaknewfilterinddH201hour.Storeat4Cin20%ethanol.