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and Critical Residues for In Vitro Biological Activity of Reteplase英文文献资料.doc

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and Critical Residues for In Vitro Biological Activity of Reteplase英文文献资料

HindawiPublishingCorporation AdvancesinHematology Volume2010,ArticleID172484,9pages doi:10.1155/2010/172484 ResearchArticle Asn12andAsn278:CriticalResiduesforInVitroBiological ActivityofReteplase NaganathMandi, KalyanaR.Sundaram, SunilK.Tandra, SumanBandyopadhyay, 1 2 3 3 andSriramPadmanabhan 4 1 2 3 4 CloneDevelopmentTeam,LupinLimited,BiotechnologyRD,Gat#1156,MulshiTaluka,GhotawadeVillage, Pune411042,India AnalyticalDevelopmentTeam,LupinLimited,BiotechnologyRD,Gat#1156,MulshiTaluka,GhotawadeVillage, Pune411042,India MammalianBioassayTeam,LupinLimited,BiotechnologyRD,Gat#1156,MulshiTaluka,GhotawadeVillage, Pune411042,India LupinLimited,BiotechnologyRD,Gat#1156,MulshiTaluka,GhotawadeVillage,Pune411042,India CorrespondenceshouldbeaddressedtoSriramPadmanabhan,srirampadmanabhan@ Received23January2010;Accepted25May2010 AcademicEditor:HennyH.Billett Copyright?2010NaganathMandietal. This is an open access article distributed under the Creative Commons Attribution License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly cited. Reteplase(rPA)isathrombolyticagentusedforthetreatmentofacutemyocardialinfarction.WestudiedtheexpressionofrPA and its selected asparagine mutants after integration into the Pichia genome. Though methanol induction of the native and therPAmutantsshowedsimilarexpressionlevels(～200–250mg/L),themutantsdisplayedsigni?cantlossofproteaseactivity. Strikingly,theclotlysisactivitiesofthesemutantswereconsiderablydi?erent.WhilemutationofAsn (N12P)oftheKringle2 12 domainshoweddelayedclotlysisactivity(t1/2 = 38min)comparedtothenativerPA(t1/2 = 33min),afasterrateofclotlysis (t1/2 =27min)wasobservedwhentheAsn (N278S)oftheserineproteasedomainwasmutated.Interestingly,theslowestclot inregulating 278 lysisactivity(t1/2=49min)demonstratedbythedoublemutant(N12P,N278S)suggeststhedominantroleofAsn 12 the?brinolyticactivityofrPA.Theresults