浙江大学学报

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1、Liuetal./JZhejiangUniv-SciB(Biomed&Biotechnol)201112(1):65-7165JournalofZhejiangUniversity-SCIENCEB(Biomedicine&Biotechnology)ISSN1673-1581(Print);ISSN1862-1783(Online)www.zju.edu.cn/jzus;www.springerlink.comE-mail:jzus@zju.edu.cnFusionexpressionofpedAgenetoobtainbiologically*activepediocinPA-1

2、inEscherichiacoli†‡Shan-naLIU,YeHAN,Zhi-jiangZHOU(SchoolofChemicalEngineeringandTechnology,TianjinUniversity,Tianjin300072,China)†E-mail:zzj@tju.edu.cnReceivedApr.26,2010;RevisionacceptedJuly23,2010;CrosscheckedDec.9,2010Abstract:Twoheterologousexpressionsystemsusingthioredoxin(trxA)asagenefusi

3、onpartinEscherichiacoliweredevelopedtoproducerecombinantpediocinPA-1.PediocinPA-1structuralgenepedAwasisolatedfromPediococcusacidilacticiPA003bythemethodofpolymerasechainreaction(PCR),thenclonedintovectorpET32a(+),andexpressedasthioredoxin-PedAfusionproteininthehoststrainE.coliBL21(DE3).Thefusi

4、onproteinwasintheformofinclusionbodyandwasrefoldedbeforepurificationbynickel-iminodiaceticacid(Ni-IDA)agaroseresincolumn.BiologicalactivityofrecombinantpediocinPA-1wasanalyzedaftercleavageofthefusionproteinbyenterokinase.Agardiffusiontestre-vealedthat512-arbitraryunit(AU)recombinantpediocinPA-1

5、wasobtainedfrom1mlculturemediumofE.coli(pPA003PED1)usingListeriamonocytogenesastheindicatorstrain.Thioredoxin-PedAfusiongenewasfurtherclonedintopET20b(+).Thioredoxin-PedAfusionproteinwasdetectedinboththeperiplasmicandcytoplasmicspaces.TherecombinantpediocinPA-1fromthesolublefractionattained384A

6、Ufrom1mlculturemediumofE.coli(pPA003PED2).Therefore,biologicallyactivepediocinPA-1couldbeobtainedbythesetwohybridgeneexpressionmethods.Keywords:Bacteriocin,Fusionexpression,Inclusionbody,PediocinPA-1,Thioredoxindoi:10.1631/jzus.B1000152Documentcode:ACLCnumber:Q7531IntroductionPediocinPA-1belong

7、stotheClassIIabacteriocinsandgainedcommercialinterestowingtoitscharac-Inthefoodindustry,foodpreservationhasal-teristicsofheatstability,highspecificactivityagainstwaysbeenanimportantaspect.SearchingnewtypesthefoodbornepathogenListe