Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens

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1、Biotechnol.Appl.Biochem.(2005)41,225231(PrintedinGreatBritain)225Cloning,expressionandcharacterizationofasialidasegenefromArthrobacterureafaciensSørenChristensen1andJanEgebjergH.LundbeckA/S,Ottiliavej9,DK-2500Valby,DenmarkSialidaseshaverecentlybeenusedintheprocessingofcloneandchar

2、acterizetheA.ureafacienssialidase.Theclinicallyrelevantasialoproteins.TheArthrobacterurea-presentpaperdescribestheisolationofanA.ureafaciensfacienssialidase(EC3.2.1.18)exhibitsbroadsubstratesialidaseexpressionclone,genesequence,characterization,specificityandisoftenusedinsuchapplic

3、ations.WerecombinantexpressioninEscherichiacoliandpurificationhaveemployedanexpressioncloningstrategytoisolateofahistidine-taggedtruncatedrecombinantformofthetheA.ureafacienssialidase.Thecloneencodesa990-A.ureafacienssialidase.Further,wedemonstratetheuseofamino-acid104kDaopen-readi

4、ng-frameproteincon-theenzymeinthedesialylationofhumanrecombinantEPOtainingthreedomains:anN-terminalcatalyticdomain,(erythropoietin).alinkerdomainwithanimmunoglobulin-likefoldandaC-terminaldomainofunknownfunction.ExpressioninExperimentalEscherichiacoliindicatesthatthesialidasepromo

5、terwasactiveinE.coli.OverexpressioninE.coliresultedStrains,mediumandchemicalsinseveraltruncatedforms.A54kDatruncatedvariantA.ureafaciensA.T.C.C.7562wasobtainedfromthewasgenerated,expressedandpurified,anditsfeasi-AmericanTypeCultureCollection,Manassas,VA,U.S.A.bilityforuseinanerythr

6、opoietindesialylationprocessThemediumforculturingA.ureafacienscontained(mg/ml):wasdemonstrated.bactotryptone,5.0;AcNeu(N-acetyl-α-D-neuraminicacid),1.0;(NH4)2HPO4,2.0;KH2PO4,1.0;NaCl,3.0;MgSO4,Introduction1.0;FeSO4,0.020(inwater)(AUmedium).Humanrecombi-nantEPOwasobtainedfromDragon

7、PharmaceuticalsSialidases(EC3.2.1.18)havebeenreportedtoactasviru-(Vancouver,BC,Canada).TEbufferwas10mMTrislencefactorsinpathogenicbacteriaandviruses.Thebacter-(pH7.5)/1mMEDTA.iumArthrobacterureafaciensisclassifiedasanon-pathogenicAssaysofsialidaseactivityorganism,butitdoesexpressas

8、ialidase,suggestingaFluorescencea