20318_ftp

analytical chemistry

CausesofProteolyticDegradationof

SecretedRecombinantProteinsProducedinMethylotrophicYeastPichiapastoris:CaseStudyWithRecombinantOvineInterferon-T

JayantaSinha,BradleyA.Plantz,MehmetInan,MichaelM.Meagher

BiologicalProcessDevelopmentFacility,DepartmentofChemicalEngineering,UniversityofNebraska-Lincoln,NE;e-mail:jsinha@unlnotes.unl.edu

Received19March2004;accepted25August2004

Publishedonline3December2004inWileyInterScience(doc.docsou.com).DOI:10.1002/bit.20318

Abstract:Itwasobservedthatduringfermentativeproductionofrecombinantovineinterferon-H(r-oIFN-H)inPichiapastoris,asecretedrecombinantprotein,theproteinwasdegradedincreasinglyafter48hofinductionandtherateofdegradationincreasedtowardstheendoffermen-tationat72h,whenthefermentationwasstopped.Proteases,whoseprimarysourcewasthevacuoles,wasfoundinincreasinglevelsinthecytoplasmandinthefermentationbrothafter48hofinductionandreachedmaximalvalueswhenthebatchwascompletedat72h.Proteaselevelsatvariouscellfractionsaswellasintheculturesupernatantwerelowerwhenglycerolwasusedasthecarbonsourceinsteadofmethanol.Itcanbecon-cludedthatmethanolmetabolismalongwithcelllysisto-wardstheendoffermentationcontributestoincreasedproteolyticactivityandeventualdegradationofrecombi-nantprotein.B2004WileyPeriodicals,Inc.

Keywords:proteolyticdegradation;secretedrecombinantinterferon-H;methanol;Pichiapastoris

INTRODUCTION

Proteolyticdegradationhasbeenaperpetualproblemwhenyeastsareemployedtoexpressrecombinantproteins(VanDenHazeletal.,1996;Kobayashietal.,2000;Gimenezetal.,2000).Yeastvacuolescontainvariousproteaseswhoselevelsvaryaccordingtothenutritionalconditions(Hansenetal.,1977).Saccharomycescerevisiaeisknowntoexpressseveralproteases,someofwhichhavebeenwellidentified(VanDenHazeletal.,1996).Incomparison,theproteasesinPichiapastorisarenotwellcharacterized,althoughtherehavebeenseveralreportsofproteolyticdegradationofre-combinantproteinsproducedinP.pastoris(Sinhaetal.,2003;Kobayashietal.,2000;ZhouandZhang,2002;Wertenetal.,1999).Afewremedialmeasureslikeadditionofcasaminoacids,additionofproteaseinhibitors,oruseofprotease-deficientstrainshavebeensuggestedasmeanstocircumventproteolyticdegradationoftherecombinantprotein(Goodricketal.,2001;Kurokawaetal.,2002;

Correspondenceto:J.Sinha

Brankampetal.,1995).However,noin-depthanalysisontheconditionsthatpromoteproteolysisorthenatureoftheproteasesactingonthedesiredproteinisexactlyknown.Therehavebeennoreportsofextracellularproteasesinyeasts,themainsourceofproteasesinSaccharomycesbeingvacuolarproteases,followedbyproteasomeandtheproteasesofthesecretorypathway(Yasuharaetal.,1994).InS.cervisiaethemajorvacuolarproteasesareproteinaseA(PrA),proteinaseB(PrB),carboxypeptidases,andamino-peptidases(VanDenHazeletal.,1996).Ithasbeenob-servedthatwhenPrAisoverexpressedinyeast,apartoftheenzymecanbesecreted(Rothmanetal.,1986).Ingen-eral,thesesolubleproteasesarecontainedwithinthevacu-oles,althoughbothvacuolarandsecretoryproteinsmovethroughendoplasmicreticulumandGolgicomplexduringtheirprocessofsynthesis(YoshihisaandAnraku,1990).Proteolyticdegradationofsecretedrecombinantproteinscanbecausedbydegradationofthecellmembraneduetocelllysisinhighcelldensityfermentation.Yeastcellsareknowntobestressedbystarvation,changeofcarbonsources,heatandpHchanges,ortoxicchemicals.Proteinsdamagedbyoxidativestressandheat-shockresponsealsoelicitaproteolyticresponse(HiltandWolf,1992).Inthisregard,proteinsdamagedbyoxidativestressareaprobability,sincemethanolmetabolismdemandshighoxygenandaby-productishydrogenperoxide.Excessproductionofthevacuolarproteasesresultedinasecretiontocytosolandeventuallytotheculturemedium(Stevensetal.,1986).Intheongoingresearch,proteaseswerefoundtoactonseveralrecombinantproteinsduringfermentation,mostprominentlyonrecombinantovineinterferon-H(r-oIFN-H).Theproteaseswerenotdetectedduringtheglycerolgrowthphaseandtheiractivityincreasedafterthefirst48hofthemethanolinductionphaseandreachedamaximumbetween60–72hoffermentation(Sinhaetal.,2003).Since,therecombinantproteinsarealsoproducedduringthemethanolinductionperiod,thepresenceofproteaseswasdeleterioustothesuccessoftheprocess.Thepurposeofthisresearchistounderstandthenatureofthe

B2004WileyPeriodicals,Inc.

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