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1、ThrombinKitsTableofContentsAbouttheKits.......................................................................................2Description2Components2ThrombinCleavage................................................................................3Smallscaleoptimization3Scale-up4Factorst
2、hataffectthrombinactivity4Monitoringcleavage5BiotinylatedThrombinCapture..................................................................5References...........................................................................................6Copyright1992–1998byNovagen,Inc.Allrightsreser
3、ved.S?Tag?,T7?Tag?,His?Bind?,pBAC?,CBD?Tag?,PerfectProtein?,theNovagenlogoandnamearetrademarksofNovagen,Inc.Orders:800526-7319TechnicalService:800207-0144TB1888/98Novagen1ThrombinKitsAbouttheKitsThrombinCleavageCaptureKit69022-3BiotinylatedThrombin69672-3Thrombin,RestrictionGrade69671-3
4、DescriptionThrombinisanendoproteasethatnaturallyfunctionsasabloodclottingfactortoconvertfibrinogentofibrin.Humanthrombinisoneofthemostactivesite-specificproteases,whichisanadvantagebecauseaverylowmassratioofenzymetotargetproteinisneededforefficientcleavage.Althoughnosingleconsensusseque
5、ncedescribesthespecificityofthrombincutting,cleavagefrequentlyoccursaftertheproline-arginineresiduepairwhenproperlyexposedinathree-dimensionalstructure.Novagen’sBiotinylatedThrombinandRestrictionGradeThrombinarefunctionallytestedforspecificcleavageofthesequenceLeuValProArgˉGlySerinrecom
6、binantfusionproteins.Thisthrombinrecognitionsequenceisencodedbymanypopularexpressionvectors,includingavarietyofNovagen’svectors.Thecleavagesiteisusuallylocatedbetweenthecloningsitesandothervector-encodedfusionpeptidesequences,whichallowsproteolyticremovalofthefusedpeptidefromtheexpresse
7、dtargetprotein.BiotinylatedThrombinissimilartoRestrictionGradeThrombin,butithasbiotincovalentlyattachedforeasyremovaloftheenzymefromcleavagereactionsusingimmobilizedstreptavidin.Boththrombinproductsarerigorouslytestedforspecificcleavageofarecombinantfusionproteinandarefunctiona
