Itisoftendifficulttoobtainsolubleandactiveproteinsfromexpressioninprokaryotes.Often,overexpressionleadstotheproductionofinclusionbodies:insolubleaggregatesofmisfoldedprotein.Theseinclusionbodiescaneasilybepurified.Howeverthesolubilizationoftheexpressedproteincanusuallyonlybeobtainedusingstronglydenaturingconditionsandamajorproblemisthentoachieveanefficientfoldingin-vitro.

Theproblemismainlyhowtolowerthedenaturantconcentrationtoallowfoldingwhileatthesametimepreventingaggregation.

Mostpublishedmethodssuggesttoeliminatethedenaturingagentusingdialysis.ThisisusuallyamistakeasdialysisexposestheproteinsolutiontoadecreasingdenaturantgrADIentoverafewhours.Soproteinswillremainexposedforanextendedperiodoftimetoanintermediatedenaturantconcentration(2to4MUreaorguanidine)wheretheyarenotyetfoldedbutnolongerdenaturedandthusextremelypronetoaggregation.Thiswasshownasearlyas1974byasetofexperimentsbyProf.Goldbergandhisteam.Inthispaper(Londonetal.Eur.J.Biochem;vol.37,pp409-415.),itwasshownthatthefoldingyieldoftryptophanasewouldreachaminimumwhenthefoldingmixwereincubatedatintermediatedenaturantconcentration(seefig.2ofthispaper).

Next,itisimportanttolimittheaggregationusingmildsolubilizingagentsduringtherefoldingsteps.Nondetergentsulfobetaines(NDSB)arewellsuitedtothispurposeandhavebeenshowntobeparticularlyefficient(VuillardetalBiochem.J,(1995).,vol.305pp337-343//Golbergetal.Foldinganddesign(1996),vol.1pp21-27,seealsohttp://www.st-and.ac.uk/~lv2/ndsb_ref2.html.

Thefollowingprotocolcanbeusedasastartingpoint

Foldingofproteinsexpressedasinclusionbodies.

1Solubilizationofproteinsfrominclusionbodies.

-ThepelletfromalitreofbacterialsUSPensionisresuspendedin20mlof:

50mMHEPES-NaOHpH7.5,0.5MNaCl,1mMPMSF,5mMDTTcontaining0.35mg/mllysozymethenincubatedfor30minat20C.

-TritonX-100isaddedtoaconcentrationof1%(vol./vol),thisisfollowedbyultrasoundsonicationbyburstsof30secfollowedbycoolinguntilthesolutionclears.

-TheextractistreatedbyDnaseIfor1hat37C,enzymeconcentration20mg/l.

-Theinclusionbodiesaresedimentedbycentrifugationat30000gfor30minat4C.

-Thepellet(inclusionbodies)iswashedtwicewithTBSorPBScontaining1%tritonX100followedbyspinningat30000gfor30minat4C.

-Thepellet(inclusionbodies)isthesolubilizedin2ml50mMHEPES-NaOHpH7.5,6MguanidineHCl,25mMDTTandleftfor1hat4C.

-Insolublematerialisremovedbycentrifugationat100000gfor10minutes.Thisisimportanttoremoveexistingaggregatesthatcanactasnucleitotriggeraggregationduringfolding.

-Determinetheproteinconcentrationandadjustto1mg/mlusing50mMHEPES-NaOHpH7.5,6MguanidineHCl,25mMDTTandproceeddirectlytofolding.

2Foldingprotocol.

ThesolubilizedproteinsaredilutedasquicklyaspossIBLe1in10intocold(4C)foldingbuffer:

50mMHEPESpH7.5,0.2MNaCl,1mMDTT,1MNDSB256(or1MNDSB201).

thefinalproteinconcentrationshouldnotexceed0.05to0.1mg/ml.Afastanefficientmixisessential.forsmallvolumesdispensingtheproteinsolutionwithaPipettedirectlyintothefoldingbufferwhilevortexingisadequate.(keepvortexingfor30secafteraddition)Forlargervolumesonecandispensetheproteinsolutionintothefoldingbufferusingasyringeundervigorous(magneticstirrer)agitation.Keepstirringfor2minutesafteraddition.Leavefor1hat4C.TheremainingguanidineandNDSBcanthenberemovedbydialysisintoanyappropriatebuffer.

3Importantnotes.

-Thekeyparametersareproteinconcentration,guanidineresidualconcentrationandtemperature.Thevaluessuggestedhereareonlystartingpoints,tooptimisefoldingyieldstheymayhavetobeoptimised.

-Measuringthesolutionturbidityisagoodindicationofproteinaggregationand,tocompareconditions,itmaybeworthtomeasurethesample"sabsorbanceat400nm.

-NDSB256DiemethylbenzylammoniumpropanesulfonateisusuallyslightlymoreefficientthanNDSB201[3(1-pyridinio)-1-propanesulfonate]butnDSB201ismuchcheaperasitcanbeobtainedfromseveralsuppliers.

-MoredetailsonNDSBon:

http://www.st-and.ac.uk/~lv2/

Referenceliston:

http://www.st-and.ac.uk/~lv2/ndsb_ref2.html

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