bySaraLindgren,01/22/2000

Purpose

Theprotocoldescribeshowtoprepareselenomethionyl(Se-Met)proteinusingaregularE.colistrain.Seleniumcanbeusedforphasedeterminationinmulti-wavelengthanomalousdiffraction(MAD)method.Se-Metcanoftenreplacemethionineresiduesinaproteinwithoutaffectingtheprotein"sproperties,thereforeproducingaproteinadvantageousforcrystalstructuresolving.Also,theX-rayabsorptionedgeofseleniumiseasilyaccessIBLebysynchrotronrADIation,makingaSe-MetcrystalidealforcollectinganomalousX-raydiffractiondata.TheSe-MetproteinscanalsobepreparedusinginsectcellsandCHOcells,whichwillbedescribedinseparateprotocols.

Materials

• LBmedia
• antibiotics(1000xconc.)
• 1MIPTG
• M9media(minimalmedia)1Liter5xM9media:(sterilefiltered)
  • 30gNa₂HPO₄or64gNa₂HPO₄-7H₂O
  • 15gKH₂PO₄
  • 5gNH₄Cl
  • 2.5gNaCl
  Diluteandautoclavebeforeuse.
• Aminoacid50xstockUseallaminoacidsEXCEPTGly,Ala,Pro,Asn,Cys,andMetataconcentrationof2mg/mlTohelpindissolvingtheaminoacids,autoclavefor10minutes.
• 20%glucose(sterilefilteredorautoclaved)
• 1MMgSO₄(sterilefiltered)
• 2MCaCl₂(sterilefiltered)
• 0.5%(w/v)ThiamineSolution(sterilefiltered)

Procedure

Day1

1. Preparea2mLdaycultureconsistingof2mLLBmedia,2uLantibiotics(1000xconc.),andasingleE.colicolony.Growat37°Callday.
2. PrepareM9stockmedia.Diluteandautoclavebeforeuse.
3. Prepareaminoacid50xStock.
4. Preparea150mLovernightcultureconsistingof150mLLB,150uLantibiotics(1000xconc.),and150uLofdayculture.Growat37°Covernight.

Day2

1. ToeachliterM9(1xconc.)add:10mL20%Glucose(sterilefilteredorautoclaved)2mL1MMgSO₄(sterilefiltered)0.05mL2MCaCl₂(sterilefiltered)0.1mL0.5%(w/v)thiaminesolution(sterilefiltered)1mLantibiotics(1000xconc.)20mLaminoacid50xStock(Ifprecipitateisseen,heatto60-70°Candshake.)
2. InoculateM9with50mLovernightcultureandgrowuntilanOD₆₀₀=0.5-0.6.(~2.0-2.5hours)
3. Add100mgthreonine,lysinehydrochloride,phenylalaninetotheculture.Add50mgleucine,isoleucine,valinetotheculture(allassolidpowders).
4. Add120mgDL-Se-Metor60mgL-Se-Mettotheculture(asasolidpowder).
5. Continuetogrowtheculturefor15minutes.
6. Inducewith1mL1MIPTG(finalconcentration=1mM).
7. Growabout6-8hours(whateverisoptimalfortheproteinofinterest).
8. Collectcellsasusualandproceedtopurificationsteps.

ExpectedResults

• Se-MetproteinwillshowslightlylargerMWthanthenativeproteininmassspectrum.
• Se-Metproteinmaybehaveslightlydifferentlyfromthenativeproteininpurificationsandcrystallization.

References

1. Doublie,S.(1997)PreparationofSelenomethionylProteinsforPhaseDetermination.MethodsinEnzymology276,523-530.
2. Deacon,AM.,EalickSE.(1999)Selenium-basedMADphasing:settingthesitesonlargerstructures.Structure,7,R161-R166
3. ProtocoloriginallyobtainedfromQingFanatDonWiley"sLab.
4. X-rayAnomalousScattering:Principles,WebTools,andRelatedLinksprovidedbyEthanA.MerrittatUniversityofWashington.