pPicZ-A-酵母质粒

UserManualpPICZA,B,andCPichiaexpressionvectorsforselectiononZeocin™andpurificationofsecreted,recombinantproteinsCat.no.V195-20Rev.Date:7July2010Manualpartno.25-0150MAN0000035iiiiiTableofContentsImportantInformation................................................................................................................................vAccessoryProducts...................................................................................................................................viiIntroduction.................................................................................................................................................1Overview.......................................................................................................................................................1Methods........................................................................................................................................................2CloningintopPICZA,B,andC...............................................................................................................2MultipleCloningSiteofpPICZA...........................................................................................................5MultipleCloningSiteofpPICZB............................................................................................................6MultipleCloningSiteofpPICZC............................................................................................................7PichiaTransformation..................................................................................................................................9ExpressioninPichia....................................................................................................................................13Purification..................................................................................................................................................15Appendix....................................................................................................................................................17Recipes.........................................................................................................................................................17Zeocin™........................................................................................................................................................19pPICZVector............................................................................................................................................21LithiumChlorideTransformationMethod.............................................................................................23ConstructionofInVitroMultimers..........................................................................................................25TechnicalSupport.......................................................................................................................................33PurchaserNotification...............................................................................................................................34References....................................................................................................................................................35ivvImportantInformationContents6μgofeachofpPICZαA,B,andCvectorinTEbuffer,pH8.0*(40μlat150ng/μl)*TEbuffer,pH8.0:10mMTris-HCl,1mMEDTA,pH8.0Shipping/StorageThevectorsareshippedonweticeandshouldbestoredat–20°C.ReferenceSourcesThepPICZαA,B,andCvectorsmaybeusedwiththeOriginalPichiaExpressionKit(Cat.no.K1710-01)andareincludedintheEasySelect™PichiaExpressionKit(Cat.no.K1740-01)availablefromInvitrogen.AdditionalgeneralinformationaboutrecombinantproteinexpressioninPichiapastorisisprovidedinthemanualsfortheOriginalPichiaExpressionKitandtheEasySelect™PichiaExpressionKit.ThemanualscanbedownloadedfromourWebsite()orobtainedbycallingTechnicalSupport(seepage33).FormoreinformationabouttheOriginalPichiaExpressionKitortheEasySelect™PichiaExpressionKit,refertoourWebsiteorcontactTechnicalSupport.MoredetailedinformationandprotocolsdealingwithPichiapastorismayalsobefoundinthefollowinggeneralreference(seepageviifororderinginformation):Higgins,D.R.,andCregg,J.M.(1998)PichiaProtocols.InMethodsinMolecularBiology,Vol.103.(J.M.Walker,ed.HumanaPress,Totowa,NJ)RecommendedPichiaHostStrainWerecommendusingtheX-33PichiastrainasthehostforexpressionofrecombinantproteinsfrompPICZα.OtherPichiastrainsaresuitable.TheX-33PichiastrainisavailablefromInvitrogen(seepageviifororderinginformation)andhasthefollowinggenotypeandphenotype:Genotype:Wild-typePhenotype:Mut+ContinuedonnextpageviImportantInformation,continuedMaterialsNeededFortheproceduresdescribedinthismanual,youwillneedthefollowingreagentsandequipment.Additionalreagentsmayberequired.Pleasecheckeachexperimenttoensureyouhaveallthereagentsnecessary.Seepagesvii–viiifororderinginformation.Equipment