High Cleavage Efficiency of a 2A Peptide Derived from Porcin

T2A是一个很好用的工具。我认为在同一个启动子下,启动两个独立蛋白表达,至少有两种选择：1. 加IRES。他在遇到stop coden时让核糖体不掉下来,从而继续翻译出两个独立的蛋白。但是,IRES有570bp,比较大。2. 加T2A。他可以在生成整条mRNA后,自剪切成两个独立的蛋白。而且,序列很短,只是一个peptide。可以参考这个文献

HighCleavageEfficiencyofa2APeptideDerivedfromPorcineTeschovirus-1inHumanCellLines,ZebrafishandMice

JinHeeKim1,2,Sang-RokLee3,4,Li-HuaLi2,6,Hye-JeongPark1,3,Jeong-HohPark1,KwangYoulLee5,Myeong-KyuKim7,BooAhnShin2*,Seok-YongChoi1*

1DepartmentofBiomedicalSciences,ChonnamNationalUniversityMedicalSchool,Gwangju,RepublicofKorea,2ResearchInstituteofMedicalSciences,ChonnamNationalUniversityMedicalSchool,Gwangju,RepublicofKorea,3DepartmentofBiology,ChosunUniversity,Gwangju,RepublicofKorea,4ResearchInstituteofKimandJungCo.Ltd.,Hwasun,RepublicofKorea,5CollegeofPharmacyandResearchInstituteofDrugDevelopment,ChonnamNationalUniversity,Gwangju,RepublicofKorea,6DepartmentofPathogenBiology,HainanMedicalUniversity,Haikou,People’sRepublicofChina,7DepartmentofNeurology,ChonnamNationalUniversityMedicalSchool,Gwangju,RepublicofKorea

Abstract

Whenexpressionofmorethanonegeneisrequiredincells,bicistronicormulticistronicexpressionvectorshavebeenused.Amongvariousstrategiesemployedtoconstructbicistronicormulticistronicvectors,aninternalribosomalentrysite(IRES)hasbeenwidelyused.DuetothelargesizeanddifferenceinexpressionlevelsbetweengenesbeforeandafterIRES,however,anewstrategywasrequiredtoreplaceIRES.Aself-cleaving2ApeptidecouldbeagoodcandidatetoreplaceIRESbecauseofitssmallsizeandhighcleavageefficiencybetweengenesupstreamanddownstreamofthe2Apeptide.Despitetheadvantagesofthe2Apeptides,itsuseisnotwidespreadbecause(i)therearenopubliclyavailablecloningvectorsharboringa2Apeptidegeneand(ii)comprehensivecomparisonofcleavageefficiencyamongvarious2Apeptidesreportedtodatehasnotbeenperformedindifferentcontexts.Here,wegeneratedfourexpressionplasmidseachharboringdifferent2Apeptidesderivedfromthefoot-and-mouthdiseasevirus,equinerhinitisAvirus,Thoseaasignavirusandporcineteschovirus-1,respectively,andevaluatedtheircleavageefficiencyinthreecommonlyusedhumancelllines,zebrafishembryosandadultmice.Westernblottingandconfocalmicroscopicanalysesrevealedthatamongthefour2As,theonederivedfromporcineteschovirus-1(P2A)hasthehighestcleavageefficiencyinallthecontextsexamined.Weanticipatethatthe2A-harboringcloningvectorswegeneratedandthehighestefficiencyoftheP2Apeptidewedemonstratedwouldhelpbiomedicalresearcherseasilyadoptthe2Atechnologywhenbicistronicormulticistronicexpressionisrequired.

Citation:KimJH,LeeS-R,LiL-H,ParkH-J,ParkJ-H,etal.(2011)HighCleavageEfficiencyofa2APeptideDerivedfromPorcineTeschovirus-1inHumanCellLines,ZebrafishandMice.PLoSONE6(4):e18556.doi:10.1371/journal.pone.0018556Editor:VolkerThiel,KantonalHospitalSt.Gallen,Switzerland

ReceivedJanuary4,2011;AcceptedMarch4,2011;PublishedApril29,2011

Copyright:ß2011Kimetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

Funding:ThisworkwassupportedbytheBasicScienceResearchProgramthroughtheNationalResearchFoundationofKorea(NRF)fundedbytheMinistryofEducation,ScienceandTechnology(2009-0071597and2009-0070204),agrantoftheKoreanHealthTechnologyR&DProject,MinistryforHealth,Welfare&FamilyAffairs,RepublicofKorea(A100402)andtheKoreaScienceandEngineeringFoundationthroughtheMedicalResearchCenterforGeneRegulation(R13-2002-013-06002-0)atChonnamNationalUniversity.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

CompetingInterests:SRLisanemployeeofKim&JungCo.Ltd.AllauthorsconfirmadherencetoallthePLoSONEpoliciesonsharingdataandmaterials.*E-mail:bashin@chonnam.ac.kr(BAS);zebrafish@chonnam.ac.kr(S-YC)

Introduction

Inbiomedicalresearch,thesimultaneousexpressionofmorethanonegeneincellsororganismsusingasingleplasmidissometimesrequired.Tothisend,severalstrategieshavebeenemployed:(i)multiplepromotersfusedtothegenes’openreadingframes(ORFs);(ii)insertionofsplicingsignalsbetweengenes;fusionofgeneswhoseexpressionsaredrivenbyasinglepromoter;(iii)insertionofproteolyticcleavagesitesbetweengenes;and(iv)insertionofinternalribosomalentrysites(IRESs)betweengenes[1,2].

Ofthesestrategies,IREShasbeenwidelyusedduetothefollowingadvantages:(i)ensuredcoexpressionofgenesbeforeandaftertheIRES;(ii)feasibilityofaddingsubcellularlocalizationsequencestothegeneafterIRES;and(iii)availabilityofcommercialexpressionplasmidsharboringIRES[2].However,IREShastwomajorlimitations[2,3].First,thesizeofIRESis

usuallylongerthan500nucleotides,whichcouldbeaproblemwhenalargeinsertisclonedintoIRES-containingviralvectorswithlimitedcloningcapacityorwhenmultipleIRESsareusedtogeneratemulticistronicplasmids.Second,translationefficiencyofageneplacedaftertheIRESismuchlowerthanthatofagenelocatedbeforeIRES.

Theselimitationscanbeovercomebya2Apeptide,a‘‘self-cleaving’’smallpeptidefirstidentifiedbyRyanandcolleaguesinthefoot-and-mouthdiseasevirus(FMDV),amemberofthepicornavirus[4].Theaveragelengthof2Apeptidesis18–22aminoacids.Thedesignation‘‘2A’’referstoaspecificregionofpicornaviruspolyproteinsandarosefromasystematicnomencla-tureadoptedbyresearchers.InFMDV,itisdelineatedatitsownC-terminusby‘cleavage’(tobemoreprecise,byribosomeskipping)andatitsN-terminusbyaproteolyticcleavageor‘trimming’fromtheupstreamcapsidprotein1Dbythe3C/3CDproteinase.Initially,itwasspeculatedthateithera

T2A是一个很好用的工具。我认为在同一个启动子下,启动两个独立蛋白表达,至少有两种选择：1. 加IRES。他在遇到stop coden时让核糖体不掉下来,从而继续翻译出两个独立的蛋白。但是,IRES有570bp,比较大。2. 加T2A。他可以在生成整条mRNA后,自剪切成两个独立的蛋白。而且,序列很短,只是一个peptide。可以参考这个文献

HighCleavageEfficiencyofaP2APeptide

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