PNGaseF,PeptideNGlycosidaseF,N-Glycosidase,N-Glycanase

RecombinantPNGaseFisisolatedfromaE.colistraincontainingacloneoftheElizabethkingiamiricolagene.Thereisnodetectabledifferenceinactivityorspecificactivityoftherecombinantenzymefromthenativeenzyme(E-PNG01).

PNGaseFcleavesasparagine-linked(N-linked)oligosaccharidesfromglycoproteins.PNGaseFdeaminatesasparaginetoasparticacid,leavingtheoligosaccharidesintact.Denaturationincreasestherateofcleavageupto100x.MostnativeproteinscanstillbecompletelyN-deglycosylatedbutincubationtimemustbeincreased.PNGaseFwillremainactiveunderincubationconditionsforatleast72hours.PNGaseFwillnotremoveoligosaccharidescontainingAlpha-(1,3)-linkedcorefucosecommonlyfoundonplantglycoproteins;forthispurpose,usepeptideN-glycosidaseA.

PNGaseFSourcerecombinantPNGaseFgenefromElizabethkingiamiricolainE.coli

EC3.5.1.52

Contents
60µlaliquotofrecombinantPNGaseF(0.3U)in20mMTris-HCl,pH7.5
Includedwith20µLand60µLpacksizes
5xPNGaseFReactionBuffer7.5forPNGaseF–250mMsodiumphosphate,pH7.5
PNGaseFDenaturationSolution–2%SDS,1MBeta-mercaptoethanol
PNGaseFTritonX-100–15%solution

SpecificActivity>25U/mg

Activity5U/ml

Molecularweight36,000daltons

pHrangeforPNGaseF6-10,optimum7.5

PNGaseFSuggestedusage
1.Addupto200µgofglycoproteintoanEppendorftube.Adjustto35µlfinalvolumewithde-ionizedwater.
2.Add10µl5xPNGaseFReactionBuffer7.5and2.5µlofPNGaseFDenaturationSolution.Heatat100˚Cfor5minutes.
3.Cool.Add2.5µlofPNGaseFTritonX-100andmix.
4.Add2.0µlofPNGaseFtothereaction.Incubate3hoursat37˚C.

SpecifictityPNGaseFcleavesasparagine-linked(N-linked)oligosaccharidesfromglycoproteins.PNGaseFdeaminatesasparaginetoasparticacid,leavingtheoligosaccharidesintact.Denaturationincreasestherateofcleavageupto100x.MostnativeproteinscanstillbecompletelyN-deglycosylatedbutincubationtimemustbeincreased.PNGaseFwillremainactiveunderincubationconditionsforatleast72hours.PNGaseFwillnotremoveoligosaccharidescontainingAlpha-(1,3)-linkedcorefucosecommonlyfoundonplantglycoproteins;forthispurpose,usepeptideN-glycosidaseA.

SpecificActivityDefinedastheamountofenzymerequiredtocatalyzethereleaseofN-linkedoligosaccharidesfrom1micromoleofRNaseBin1minuteat37˚C,pH7.5.CleavageismonitoredbySDS-PAGE(cleavedRNaseBmigratesfaster).

StorageStoreenzymeat4˚C.

PNGaseFReferences
–Bayer,E.A.,F.DeMeester,T.KulikandM.Wilchek.Preparationofdeglycosylatedeggwhiteavidin.ApplBiochemBiotech53:1-9(1995)

–Elder,J.H.andS.Alexander.endo-b-N-AcetylglucosaminidaseF:endoglycosidasefromFlavobacteriummeningosepticumthatcleavesbothhigh-mannoseandcomplexglycoproteins.ProcNatlAcadSciUSA79:4540-4544(1982)

–Tarentino,A.L.,C.M.GomezandT.H.Plummer,Jr.Deglycosylationofasparagine-linkedglycansbypeptide:N-glycosidaseF.Biochemistry24:4665-4671(1985)

–TarentinoA.L.andT.H.Plummer.Enzymaticdeglycosylationofasparagine-linkedglycans:purification,properties,andspecificityofoligosaccharide-cleavingenzymesfromFlavobacteriummeningosepticum.MethEnzymol230:44-57(1994)

–TrimbleR.B.andA.L.Tarentino.Identificationofdistinctendoglycosidase(endo)activitiesinFlavobacteriummeningosepticum:endoF1,endoF2andendoF3.EndoF1andendoHhydrolyzeonlyhighmannoseandhybridglycans.JBiolChem266:1646-1651(1991)

–Taga,E.M.,A.WaheedandR.L.VanEtten.Structuralandchemicalcharacterizationofahomogeneouspeptide-N-glycosidasefromalmond.Biochemistry23:815-22(1984)

–TarentinoAL,TrimbleRB,PlummerTH.Enzymaticapproachesforstudyingthestructure,synthesis,andprocessingofglycoproteins.MethodsinCellBIOLOGy:32:111–39(1989)

AnthonyL.,TarentinoandThomasH.PlummerJr.Enzymaticdeglycosylationofasparagine-linkedglycans:Purification,properties,andspecificityofoligosaccharide-cleavingenzymesfromFlavobacteriummeningosepticum.MethodsinEnzymology:230:44-57.(1994)

–TarentinoAL,PlummerTH.Oligosaccharideaccessibilitytopeptide:N-glycosidaseaspromotedbyprotein-unfoldingreagents.TheJournalofBiologicalChemistry.257(18):10776–80.(1982)

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