Pichiapastorisisamethylotropicyeastusedtoexpresshighamountsofprotein.Secretedexpressionisachievedwithacleavalablefaktor.Toyieldhighexpressionstrainsitisimportanttoselectformultiplecopiesoftheinsertinthepichiagenome.ThisisachievedwithselectiononhighZeocineconcetrationplates(2g/ml).

Material:

• GlycerolorYPDagarplatecontainingPichiapastoris
• YPDMedium(YeastPeptoneDextrose)600ml
• Zeocine(stocksolution100mg/ml)0.6ml
• BMMYmedium(BufferedMethanolcomplexmedium)600ml
• anti-foamagent
• autoclaved2lflasks

Procedure:

StrikeoutsomeglycerolonaYPD+0.1mg/mlZagarplate.Pickuponecolonyandstarttogrowtheculture.

1.Growingculture

• Inoculateacloneinasterile2lflaskcontaining600mlYPD+0.1mg/mlZeocine.Useanti-foamagentifnecessary.
• Shaketheculturefor2daysat30°Cand250rpm

(thecolorofthecultureshouldbewhiteandtheODshouldbe>10).2.Induction

• Centrifugethecultureat3000gfor5min.
• ResUSPendthepelletin500mlBMMY(containing1%MeOH)ina2lflask.

3.Expression

• Shakeat30°C250rpmfor3-5days.Useanti-foamagentifnecessary.
• Add1%MeOHtothecultureevery24h

(DuringtheexpressionthecolorofthecultureiswhiteandtheODreaches60).4.Purification

• PurifythesupernatantfollowingtheprotocolforthepurificationofE.coliderivedL19scFv.

Mediarecipes:

YPD:1%yesatextract2%peptone2%dextrose2%Agarifagarplatesareneeded

Dissolve10gyeastextractand20gpeptonein900mlwater(20gagarifneeded).Autoclavefor20minonliquidcycle.Add100ml20%dextrose

(addZeocineifnecessary)

BMMY:1%yeastextract2%peptone100mMpotassiumphosphatebufferpH6.01.34%YNB(yeastnitrogenbase)4x10-5biotin1%methanol

Dissolve10gyeastextractand20gpeptonein700mlwater.Autoclave20minonliquidcycle.Cooltoroomtemperaturethenaddthefollowingandmixwell:

100ml1MpotassiumphophatebufferpH6100mlYNB10X2ml500Xbiotin100ml10%MeOHAttention:Zeocinehasashelflifeofapproximately2weeksat4°CBacterialZeocineselectionrequireslowsaltconcentration