蛋白质相互作用实验技术的最新进展

HEREDITAS(Beijing)201311,35(11):1274―1282ISSN0253-9772综述收稿日期:2013−06−05;修回日期:2013−08−11基金项目:(31171615,31171543)作者简介:,,E-mail:[e-mail]通讯作者:,,,E-mail:[e-mail]网络出版时间:2013-9-211:50:29URL::10.3724/SP.J.1005.2013.01274蛋白质相互作用实验技术的最新进展王明强1,武金霞1,张玉红2,韩凝1,边红武1,朱睦元11.,310058;2.,850000蛋白质相互作用的研究,是揭示生物体正常生长发育及其应对各种生物或(和)非生物胁迫的分子机制及其调控网络的重要途径。文章综述了近年来发展起来的研究蛋白质相互作用的常用实验性方法,如酵母双杂交系统、串联亲和纯化、免疫共沉淀、GSTPull-down、双分子荧光互补、荧光共振能量转移、表面等离子共振分析,介绍了其原理、发展进程,并分析了其优缺点。蛋白质相互作用;酵母双杂交系统;串联亲和纯化;免疫共沉淀;GSTPull-down;双分子荧光互补;荧光共振能量转移;表面等离子共振分析Recentadvancesinthetechniquesofprotein-proteininteractionstudyWANGMing-Qiang1,WUJin-Xia1,ZHANGYu-Hong2,HANNing1,BIANHong-Wu1,ZHUMu-Yuan11.InstituteofGenetics,CollegeofLifeSciences,ZhejiangUniversity,Hangzhou310058,China;2.TibetAcademyofAgriculturalandAnimalHushandrysciences,Lhasa850000,ChinaAbstract:Protein-proteininteractionsplaykeyrolesinthedevelopmentoforganismsandtheresponsetobioticandabioticstresses.Severalwet-labmethodshavebeendevelopedtostudythischallengingarea,includingyeasttwo-hybridsystem,tandemaffinitypurification,Co-immunoprecipitation,GSTPull-down,bimolecularfluorescencecomplementation,fluorescenceresonanceenergytransferandsurfaceplasmonresonanceanalysis.Inthisreview,wediscusstheoreticalprin-ciplesandrelativeadvantagesanddisvantagesofthesetechniques,withanemphasisonrecentadvancestocompensateforlimitations.Keywords:protein-proteininteractions;yeasttwo-hybridsystem;tandemaffinitypurification;Co-immunopreci-pitation;GSTPull-down;bimolecularfluorescencecomplementation;fluorescenceresonanceenergytransfer;surfaceplasmonresonanceanalysisDNARNA(),,11:1275[1,2],1,,1.1(Yeasttwo-hybridsystem,Y2H)FieldsSong1989[3]Gal4(Transcriptionalactivationdomain,AD)DNA(DNAbindingdomain,BD)ADBD,,ADBD,[3](1),[4];,[5],,,,,[6,7]Luo[8]p53GAL4DNASV40TVP16,,p53SV40T,图1酵母双杂交系统检测蛋白质相互作用示意图1276HEREDITAS(Beijing)201335SplitTEV,[9][10](Proteininteractome),[11,12][13][14][15][16]1.2(Tandemaffinitypurification,TAP)Rigaut[20]1999TAP。此后,TAP[17~19]TAPProteinATEV(Calmodulin-bindingpeptide,CBP)[20](2a)TAP,TAPProteinAIgG,,TEVIgG,CBP(Calmodulinbeads),,[20](2c),TAP,TAP;,TAPTAP,,[21],[22],Tagwerker[23]2006TAPHB,HBRGSH66×HisBIO3,8M6M,Ni2+-Sepharosestreptavidin-agaroseHB;HB,258,SH-TAPFlag-HA,,TAP1TAP1.3(Co-immunoprecipitation,Co-IP),,(2,bd)(FlagHAc-mycProteinAGFP6×His),,Co-IP,GFP(27kDa),[33~35],Co-IP,表1常用TAP标签及其评价(kDa)Tag-1Tag-2AC-TAP20ProteinACBPTEVTAPtag[20]GS-TAP19ProteinGSBPTEVtag[24,25]LAP36EGFPS-peptide/6xHISTEV/HR3CEGFP[26]SF-TAP5StrepIIFLAG-,[27]SH-TAP5SBPHA-,[28]SPA83×FlagCBPTEV,[29]Flag-HA3FlagHA-,[30]CHH14CBP6×His3×HA-3,[31]TAPa26ProteinA9×myc/6×HisHR3CHR3C4[32]HB611RGS6H6×HisBIOHTEV,[23]11:1277图2TAP、Co-IP融合蛋白结构和TAP、Co-IP流程示意图(a)AC-TAP;(b)Flag;(c)TAP;(d)Co-IPRothbauer[36]13kDaGFP(GFP-bindingpeptide,GBP)N--(N-hydroxysuccinimide-sepharose),GFP-nano-trap;GFP-nano-trapGFP[37][38][39][40]GFP,1278HEREDITAS(Beijing)201335Co-IPTAP,,,,,,Co-IP,,Co-IPBiFCFRET[2,41]1.4GSTPul-downGSTPull-downGSTPull-down(Baitprotein)GST(Glutathione-S-transferase),GST,,[42]Co-IP,GSTPull-down,,,;GSTPull-down,GST,[43]1.5(Bimolecularfluorescencecomplementation,BiFC)(Bimolecularfluorescencecom-plementation,BiFC),,baitprey,baitprey,[44,45](3)Ghosh[45]GFPBiFCHu[46]EYFPBiFC,N-terminalGFP-S65T+C-terminalCFP[47]mRFP1-Q66T[48]mCherry[49][50,51]BiFC-FRETBiFCFRET,3[52]BiFC,,,,,[44~46]BiFC,,,[46]BiFC图3BiFC检测蛋白质相互作用示意图1.6(Fluorescenceresonanceen-ergytransfer,FRET)(Fluorescenceresonanceen-ergytransfer,FRET),Förster1948[53],FRETbaitprey(ECFP)(EYFP),baitprey,(10nm),[54,55](4)BFP-GFPBFP-EGFP11:1279CFP-YFP[56]FRET,;;,[57];,,FRET(40%),[58];,图4FRET检测蛋白质相互作用示意图1.7(Surfaceplasmonreso-nanceanalysis),,[59]SPR3SPR(),,,,[60][61];[60]2,,,,,参考文献(References):[1]HuZJ.Analysisstrategyofprotein-proteininteractionnetworks.MethodsMolBiol,2013,939:141–181.[DOI][2]DeLasRivasJ,FontanilloC.Protein-proteininteractionnetworks:unravelingthewiringofmolecularmachineswithinthecell.BriefFunctGenomics,2012,11(6):489–496.[DOI][3]FieldsS,SongOK.Anovelgeneticsystemtodetectprotein-proteininteractions.Nature,1989,340(6230):245–246.[DOI][4]MillerJP,LoRS,Ben-HurA,DesmaraisC,StagljarI,NobleWS,FieldsS.Large-scaleidentificationofyeastintegralmembraneproteininteractions.ProcNatlAcadSciUSA,2005,102(34):12123–12128.[DOI][5]NatsumeT,NakayamaH,JanssonÖ,IsobeT,TakioK,MikoshibaK.Combinationofbiomolecularinteractionanalysisandmassspectrometricaminoacidsequencing.AnalChem,2000,72(17):4193–4198.[DOI][6]KannoA,OzawaT,UmezawaY.Detectionofprotein-proteininteractionsinbacteriabyGFP-fragmentreconsti-tution.MethodsMolBiol,2011,705:251–258.[DOI][7]SuterB,KittanakomS,StagljarI.Two-hybridtechno-logiesinproteomicsresearch.CurrOpinBiotechnol,2008,19(4):316–323.[DOI][8]LuoY,BatalaoA,ZhouH,ZhuL.Mammaliantwo-hybridsystem:acomplementaryapproachtotheyeasttwo-hybridsystem.Biotechniques,1997,22(2):350–352.[DOI][9]WehrMC,LaageR,BolzU,FischerTM,GrnewaldS,ScheekS,BachA,NaveKA,RossnerMJ.Monitoringregulatedprotein-proteininteractionsusingsplitTEV.NatM