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GST Fusion Protein Purification from Yeast

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GSTFusionProteinPurificationfromYeast

5mlovernightcultureofyourfavoriteyeastinyourfavoritemedium.
Inoculate50mlandgrow30oCshakingO/NuntilOD600=0.8to1.2.ForSCDculturesuse1/500and1/1500dilutions.
Optional:addalpha-factorto2.5µM.Continueshakingat30oCfor60min.
Add1MNaN3to10mM(finalconcentration)andmoveculturestoice.Everythingmustremaincoldfromhereonout.
Spincells3K,10minat4oC.
DiscardsupeandresUSPendcellswith0.5ml10mMNaN3.Transferto1.5mlmicrofugetube(notautoclaved).
Spin3K,10minat4oC.Alternativelyspin8K,1minatroomtemperature.
Discardsupe.Optional:freezepelletat-80oC.
Resuspendin1mlofcold10mMNaN3.
MeasureOD600.Adjustvolumessothatthereareanequalnumberofcellsineachsample.AtotalOD600of30persampleisbest.
Washwith1mlofLysisBuffer.
Spin3K10minat4oCanddiscardsupe.
Resuspendin400ulLysisBuffer.
Addascoopofglassbeadstoa0.5mlPCRtube.TransfercelllysatetothePCRtube
Vortex1min,4X.Keepsamplescoldbetweenvortexing.
Pokeaholeinthebottomofthetubeandspincelllysateinanewmicrofugetube1.5Kor500Xg,10min.,4oC.
Transferliquidfrombottomtubeintoanewmicrofugetube.
Spinagain1.5K,10min,4oCandagaintransferliquidintoanewmicrofugetube.
AddTritonX-100to1.5%androckfor60minat4oC.
Spin(3K,10min,4oC)andtransferthesupetoanewmicrofugetube.
Remove30ulofliquidandadd30ul2XSDSPAGESamplebuffer.ThiswillreflectproteincontentbeforeGlutathionepurification.
Totheremainingliquid,add100ul40%slurryofGlutathionebeadsandmixat4oCfor2h(overnightisusuallyfine).Glutathionebeadsshouldbeprewashed3XwithPBSand1Xwithlysisbufferbeforeresuspendingasa40%slurryinlysisbuffer.
WashglutathionebeadsfivetimeswithPBS,1%TritonX-100,300mMNaClatRT.Spin2K,5min,atroomtemperature.Rocksamplefor5minbetweenwashes.Changetubesafterthefirstwashtoreducenonspecificbindingtothetubeitself.
ResuspendinSDS-PAGESamplebuffer.
- Alternativelyelute3timeswith1-2columnvolumesof5-10mMreducedglutathione,50mMTrispH8,andmixwith6XSDS-PAGEsamplebufferbeforestrippingthebeadswithSDS-PAGEsamplebuffer.
Heatto100oCfor10min.Thenstoreat-20oC.ProteinisreadytoberunonSDS-PAGEGel.

LysisBuffer(20ml)

Stock	Volume	Final
3.3M*Triethanolamine(pH7.2)	194µl*	40mM
0.5MEDTA(pH8)	80µl	2mM
5MNaCl	600µl	150mM
0.1MDTT	400µl	2mM
10mMAEBSF	0.4ml	0.2mM
1.5mg/mlleupeptin	200µl	15µg/ml
0.5mg/mlpepstatin	20µl	20µg/ml
1Mbenzamidine	20µl	1mM
0.5mg/mlaprotinin	400µl	10µg/ml
100mMb-glycerolphosphate	20µl	100µM
50mMNa-o-vanadate	200µl	0.5mM
	HOHto20mls

NOTES

ThankstoPaulDiBelloandJiyoungChafortheirrefinementsofthisprotocol.

*Indicatesacorrectionfromanearilerversionoftheprotocol.

1.ForlysisinthepresenseofGDPandGTPIuseafinalconcentrationof10uMGDPor20uMGTPgammaSandafinalconcentrationof3mMMgCl2inthelysisbuffer.

2.Youcansubstituteproteaseinhibitorcocktail(SigmaP8215)forindividualproteaseinhibitors(AEBSF,leupeptin,pepstatin,benzamidine,aprotinin).3.Forlysistodeterminephosphorylation,youmaywishtoaddmorephosphataseinhibitors(inadditiontobeta-glycerolphosphateandNa-o-vanadate)tothelysisbuffer,oryoumaywishtoomitphosphataseinhibitorsaltogether:

50mMNa-M-Vanadate	200ml	0.5mM
100mMNa-pyrophosphate	2ml	10mM
2mg/mlPhosvitin	10µl	1µg/ml

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GST&nbsp;Fusion&nbsp;Protein&nbsp;Purification&nbsp;from&nbsp;Yeast

GST Fusion Protein Purification from Yeast