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Two-hybridtechniqueLinglingLUBEIJINGINSTITUTEOFBRAINDISORDERS,CAPITALMEDICALUNIVERSITYTel:E-mail:

掌握雙雜交技術(shù)原理1

掌握雙雜交技術(shù)的應(yīng)用2

了解雙雜交技術(shù)的分類(lèi)及操作流程3

了解試驗(yàn)過(guò)程中可能遇到的問(wèn)題及解決辦法4教學(xué)大綱ContentsSection1PrincipleofTwo-HybridTechnique-Methodsforproteininteraction-Principleoftwohybridsystem-ClassificationoftwohybridsystemSection2ApplicationofTwo-HybridTechnique-ThegeneralstepsofTwo-Hybridsystem-ApplicationandperspectivesofTwo-HybridTechniqueWantistranscriptionalfactor?Transcriptionfactor轉(zhuǎn)錄因子(transcriptionfactor)是一群能與基因5`端上有特定序列專(zhuān)一性結(jié)合,從而保證目的基因以特定的強(qiáng)度在特定的時(shí)間與空間表達(dá)的蛋白質(zhì)分子。轉(zhuǎn)錄因子的特點(diǎn)DNA結(jié)合結(jié)構(gòu)域(DNA-bindingDomain,BD)轉(zhuǎn)錄激活結(jié)構(gòu)域(transcription-activatingdomain,AD)一般認(rèn)為,如果某個(gè)蛋白是體外轉(zhuǎn)錄系統(tǒng)中起始RNA合成所必需的,它就是轉(zhuǎn)錄復(fù)合體的一部分。根據(jù)結(jié)構(gòu)和功能的不同,可將轉(zhuǎn)錄因子分為兩個(gè)部分:轉(zhuǎn)錄激活域一般由30~100氨基酸殘基組成,這些結(jié)構(gòu)域有富含酸性氨基酸的α-螺旋

、富含Gln和富含Pro等不同種類(lèi)。一般由60~100個(gè)氨基酸殘基組成的幾個(gè)亞區(qū)組成。與轉(zhuǎn)錄因子結(jié)合的DNA區(qū)常是一段反向重復(fù)序列,因此許多轉(zhuǎn)錄因子常以二聚體形式與DNA結(jié)合。轉(zhuǎn)錄因子的DNA結(jié)合結(jié)構(gòu)域有鋅指和螺旋-轉(zhuǎn)角-螺旋等形式Homeodomain(同源域),最早來(lái)自控制軀體發(fā)育的基因,長(zhǎng)約60個(gè)氨基酸,其中的DNA結(jié)合區(qū)與helix-turn-helixmotif相似,人們把該DNA序列稱(chēng)為homeobox。主要與DNA大溝相結(jié)合。DNA結(jié)合結(jié)構(gòu)域基序

堿性螺旋--環(huán)--螺旋(basichelix-loop-helix)

該調(diào)控區(qū)長(zhǎng)約50個(gè)aa殘基,同時(shí)具有DNA結(jié)合和形成蛋白質(zhì)二聚體的功能,其主要特點(diǎn)是可形成兩個(gè)親脂性α-螺旋,兩個(gè)螺旋之間由環(huán)狀結(jié)構(gòu)相連,其DNA結(jié)合功能是由一個(gè)較短的富堿性氨基酸區(qū)所決定的。DNA結(jié)合結(jié)構(gòu)域基序GeneTranscription雙雜交技術(shù)是如何設(shè)計(jì)實(shí)施的?TheTwo-HybridSystemPrincipleGAL4UASPromoterReportergeneTranscriptionactivatorismodularDNAbindingdomain(DNA結(jié)合結(jié)構(gòu)域,DNA-BD)andactivationdomain(轉(zhuǎn)錄激活結(jié)構(gòu)域,AD)DNA-BDADTranscription酵母雙雜交原理

典型的真核生長(zhǎng)轉(zhuǎn)錄因子都含有二個(gè)不同的結(jié)構(gòu)域:DNA結(jié)合結(jié)構(gòu)域和轉(zhuǎn)錄激活結(jié)構(gòu)域。前者可識(shí)別DNA上的特異序列,并使轉(zhuǎn)錄激活結(jié)構(gòu)域定位于所調(diào)節(jié)的基因的上游,轉(zhuǎn)錄激活結(jié)構(gòu)域可同轉(zhuǎn)錄復(fù)合體的其他成分作用,啟動(dòng)它所調(diào)節(jié)的基因的轉(zhuǎn)錄。二個(gè)結(jié)構(gòu)域不但可在其連接區(qū)適當(dāng)部位打開(kāi),仍具有各自的功能。而且不同兩結(jié)構(gòu)域可重建發(fā)揮轉(zhuǎn)錄激活作用。酵母雙雜交系統(tǒng)利用雜交基因通過(guò)激活報(bào)道基因的表達(dá)探測(cè)蛋白-蛋白的相互作用。主要有二類(lèi)載體:a含DNA-bindingdomain的載體;b含DNA-activatingdomain的載體。1.SystembasedonGal4TheDNA-BD/proteinX(bait)bindspromoterbutcannotactivatetranscription:AD/libraryfusionproteincannotbindtothepromoteraloneandthuscannotactivatetranscription:ABInteractionbetweenthebaitandlibraryproteinsinvivoactivatestranscriptionofthereportergene:CApplicationsIdentificationofnewproteinsandelucidatesbiologicalpathwaysIdentificationofaminoacidsorproteindomainsrequiredforprotein-proteininteractionsSimpleidentificationandisolationofgenesencodingproteinsinvolvedinprotein-proteininteractionsStudyofsignaltransduction,cellgrowthanddifferentiation,geneexpression,secretionandmetabolismSystembasedonRNApolIII1997,Marsoliteretal.Reportergene:SNR6SNR6promotorwasmodifiedtohaveaGal4bingdingdomain.ThisisconvinientforGal4fusedproteintobindonSNR6promoter.Preyproteinfusedwithτ138sub-unitofTFIIICfactor,τ138sub-unitcanrecruitmentpolIIISNR6wastranscripted.ApplicationtostudytheproteinsthatthemselvesarepolIItranscriptionactivatedfactorReversetwo-hybridsystemPrinciple:constructareversereportergene.cellscannotsurvivewhenreportergenewasexpressed。Reportergene:URA3(編碼乳清苷5-磷酸脫羧酶,基因產(chǎn)物為尿嘧啶合成所必需,但能將5-氟乳清酸轉(zhuǎn)化為一種有毒物質(zhì)。)Selectmedium:add5-FOAApplication:可以從大量的突變體中直接篩選出導(dǎo)致蛋白質(zhì)間相互作用減弱或消失的突變位點(diǎn)Split-hybridsystemAnotherreversesystem。利用了兩個(gè)相互整合的報(bào)道基因,第一個(gè)報(bào)道基因含有大腸桿菌tetR因子的編碼序列,其表達(dá)受上游lexA操縱子調(diào)控;第二個(gè)報(bào)道基因是His3,啟動(dòng)子區(qū)含有tet操縱子序列。分別與BD和AD融合的兩個(gè)蛋白質(zhì)在酵母細(xì)胞核內(nèi)如果發(fā)生相互作用,將激活tetR(四環(huán)素抑制蛋白)的表達(dá),tetR和tet操縱子序列的的結(jié)合進(jìn)一步影響His3基因的表達(dá),只有不能發(fā)生相互作用的蛋白質(zhì)才能用這個(gè)系統(tǒng)在組胺酸缺陷培養(yǎng)基上生長(zhǎng)。應(yīng)用:除了篩選突變株獲得蛋白質(zhì)結(jié)合的信息外,還可以用于發(fā)現(xiàn)可導(dǎo)致相互作用的蛋白質(zhì)間解離的藥物或其他小分子物質(zhì)。對(duì)于治療疾病非常有意義。SOSrecruitmentsystem-細(xì)胞質(zhì)內(nèi)的雙雜交系統(tǒng)-利用RAS信號(hào)轉(zhuǎn)導(dǎo)途徑的性質(zhì)。酵母細(xì)胞需要RAS信號(hào)才能生存。RAS的功能依賴(lài)于激活鳥(niǎo)苷酸交換因子(GEF),它把無(wú)活性GDP-RAS轉(zhuǎn)變成激活的GTP-RAS。酵母中天然的GEF是CDC25,它存在溫度敏感的突變體。利用人的GEFhSOS蛋白可以代替溫度敏感性突變酵母的RASGEF,即CDC25,使酵母在36℃可以存活。但是hSOS蛋白必須定位到細(xì)胞膜上才能刺激酵母RAS的鳥(niǎo)苷酸的交換。實(shí)際應(yīng)用中,一種蛋白質(zhì)和hSOS蛋白融合,另一種蛋白質(zhì)和Src豆蔻酰化膜定位信號(hào)融合,如果兩種蛋白質(zhì)發(fā)生相互作用,就會(huì)將hSOS招募到細(xì)胞膜上,從而激活RAS,使細(xì)胞在限制溫度下存活和增殖。-適合分析受體與配體相互作用SOSrecruitmentsystem細(xì)胞質(zhì)內(nèi)的雙雜交系統(tǒng)研究膜相關(guān)蛋白利用RAS信號(hào)轉(zhuǎn)導(dǎo)途徑的性質(zhì)Thesplit-ubiquitinmembraneyeasttwo-hybridsystemprovidesanalysisofproteininteractionsatthecellmembrane.BaitproteinsarefusedwiththeC-terminusofubiquitin(Cub)andatranscriptionfactor.PotentialinteractingproteinsarethencoupledtotheN-terminusofubiquitin(Nub).Ifthebaitandpreyproteinsinteractatthemembrane,thenubiquitinisreconstituted,triggeringreleaseofthetranscriptionfactortoactivatethereportergenes.Reportergene:transcriptionfactororproteinscapableofdetectionSplit-ubiquitinsystemUSPSUbiquitin-SpecificProteasesSystem,USPS:泛素專(zhuān)一性蛋白酶系統(tǒng)泛素的功能特點(diǎn),泛素有獨(dú)立的N端和C端兩部分構(gòu)成,當(dāng)分離的兩部分靠近時(shí),泛素轉(zhuǎn)移性的蛋白酶就會(huì)識(shí)別泛素,導(dǎo)致連接在其C端的蛋白解離釋放。Split-ubiquitinsystem報(bào)道基因采用轉(zhuǎn)錄激活因子來(lái)激活報(bào)道基因的轉(zhuǎn)錄或其他易被檢測(cè)到信號(hào)的蛋白質(zhì)。Split-ubiquitinsystemAdvantages:蛋白質(zhì)相互作用水解釋放報(bào)道蛋白,這樣報(bào)道蛋白如果是轉(zhuǎn)錄因子,就更容易進(jìn)入核內(nèi),活化轉(zhuǎn)錄。報(bào)道蛋白可以選用其他的蛋白質(zhì)如酶,這樣可以通過(guò)分析酶的活性來(lái)分析蛋白質(zhì)之間的相互作用。OnehybridsystemIntheonehybridsystem,proteinscapableofselectivelybindingtoaspecificDNAsequencecanbeidentifiedbyscreeningalibraryofrandomcDNA-AD.Additionally,theone-hybridsystemcanbeusedtostudytheeffectsofmutations,ligands,andregulatoryproteinsonthefunctionofaparticulartranscriptionalactivation.One-hybridsystemYeastThree-HybridSystem

研究RNA或小分子配基和蛋白質(zhì)的相互作用ClassificationAccordingtotheprinciple:RNA聚合酶II以RNA聚合酶III為基礎(chǔ)的雙雜交體系reversetwo-hybridsystem反向雙雜交系統(tǒng)Split-hybridsystem分離雜交系統(tǒng)SOSrecruitmentsystemSOS招募系統(tǒng)Split-ubiquitinsystem分離的泛素系統(tǒng)Onehybridsystem單雜交系統(tǒng)Threehybridsystem三雜交系統(tǒng)Accordingtothevector:Conventionalyeasttwo-hybridsystemMammaliantwo-hybridsystemBacteriomatchtwo-hybridsystem酵母雙雜交體系的新發(fā)展篩選載體文庫(kù)接合驗(yàn)證1.酵母菌株的多重篩選機(jī)制

位于報(bào)告基因上游的啟動(dòng)子是決定報(bào)告基因表達(dá)靈敏性和特異性最重要的因素,較早的雙雜交系統(tǒng)雖然采用了雙重報(bào)告基因,如HIS3和LacZ,但它們是受同一啟動(dòng)子的調(diào)控,具有相同的17個(gè)堿基的BD識(shí)別序列,能激活其中之一者很有可能在另一檢測(cè)中亦呈陽(yáng)性。目前設(shè)計(jì)的酵母菌株往往具有不同啟動(dòng)子調(diào)控下的報(bào)告基因。如PJ69-4A[14]具有三個(gè)報(bào)告基因:GAL1-HIS3、GAL2-ADE2和GAL7-LacZ,GAL1、GAL2、GAL7都有可被GaL4BD識(shí)別的特異序列,且彼此之間各不相同,使三種報(bào)告基因能同時(shí)產(chǎn)生假陽(yáng)性反應(yīng)的機(jī)率明顯下降。其中GAL2-ADE2,經(jīng)對(duì)照實(shí)驗(yàn)檢測(cè)為一種靈敏性、特異性均強(qiáng)的報(bào)告基因,清除假陽(yáng)性很有效。2.表達(dá)型載體的構(gòu)建也正進(jìn)行著幾方面的改進(jìn)首先,融合蛋白被置于一些可被誘導(dǎo)的啟動(dòng)子調(diào)控之下,使蛋白質(zhì)在酵母的表達(dá)量能依需要進(jìn)行調(diào)控。其次,BD或AD與作用蛋白質(zhì)的連接方式也不再只是BD或AD的C端與蛋白質(zhì)的N端,因?yàn)榈鞍踪|(zhì)的N端和其活性也有很大關(guān)系,對(duì)于常規(guī)連接找不到作用蛋白的對(duì)象,改用N端也許能有不同的發(fā)現(xiàn)。雙雜交體系中不同載體原來(lái)均采用氨芐抗性作為選擇標(biāo)志,現(xiàn)在可被改進(jìn)成具有耐氨芐、卡那霉素或氯霉素等不同耐藥基因的載體,使得原來(lái)繁瑣的從酵母中提取出單一質(zhì)粒的工作變得輕松快捷。3.用于雙雜交篩選的文庫(kù)

用于雙雜交篩選的文庫(kù)不但應(yīng)有代表每一基因的足夠豐富的多樣性,而且由于蛋白質(zhì)間發(fā)生作用的位點(diǎn)可能為蛋白質(zhì)多肽鏈的任意位置,所以對(duì)每一基因而言,還應(yīng)當(dāng)提供盡可能多地與AD連接位點(diǎn)。

插入片段不宜過(guò)大,否則因?yàn)榉翘禺惤Y(jié)合所導(dǎo)致的假陽(yáng)性也會(huì)明顯增多。選用更適宜的限制性?xún)?nèi)切酶以構(gòu)建適用于雙雜交體系的文庫(kù)是綜合提高雙雜交技術(shù)的一個(gè)重要方面。4.酵母接合

鄰近存在的兩個(gè)不同交配型的酵母配子體可能交配形成二倍體。利用這一特點(diǎn),酵母接合(yeastmating)可以很方便地將兩種不同的質(zhì)粒轉(zhuǎn)入同一酵母菌株。

目前此方法已成為酵母轉(zhuǎn)化的主要替代方法,極大的提高了可以被同時(shí)檢測(cè)的蛋白質(zhì)數(shù)量。5.體外驗(yàn)證

體內(nèi)進(jìn)行的雙雜交檢測(cè)往往需要體外的其他方法來(lái)驗(yàn)證,為了使后續(xù)的采用其他方法再次鑒別蛋白質(zhì)相互作用的步驟更易實(shí)施,有報(bào)道將雙雜交篩出的基因片段插入一個(gè)帶有抗原決定簇標(biāo)記的質(zhì)粒載體,該載體可在哺乳動(dòng)物細(xì)胞中高水平表達(dá)被抗原決定簇所標(biāo)記的蛋白質(zhì)。用BD-X質(zhì)粒和帶有AD-Y插入的抗原決定簇質(zhì)粒載體共轉(zhuǎn)染哺乳動(dòng)物細(xì)胞,即可直接進(jìn)行免疫共沉淀檢測(cè)。應(yīng)用這一方法已證實(shí)了Bcl-XL和Bad/Bax之間的相互作用。哺乳動(dòng)物雙雜交系統(tǒng)

CheckMate?

PROMEGA

●可在所選細(xì)胞系中研究相互作用,蛋白質(zhì)更可能處于其本來(lái)構(gòu)型。更好地保持了諸如糖基化、磷酸化和?;确g后的修飾?!窨焖偎矔r(shí)檢測(cè):轉(zhuǎn)染后兩天即可得到結(jié)果,而酵母系統(tǒng)則需3-4天。●穩(wěn)定轉(zhuǎn)染:pACT載體含有新霉素磷酸轉(zhuǎn)移酶基因,可用于穩(wěn)定轉(zhuǎn)染的選擇Mammaliantwo-hybridassaykit(k1602-1)細(xì)菌雙雜交基本原理

與酵母雙雜交的原理相似。通過(guò)將所要研究的蛋白質(zhì)分別與DNA結(jié)合域(DBD)與活化域(AD)融合,利用相互作用蛋白質(zhì)提供的橋聯(lián)功能,使活化域與DNA結(jié)合域結(jié)合,從而調(diào)控報(bào)告基因的表達(dá)。報(bào)告基因表達(dá)的調(diào)控結(jié)果可通過(guò)生化或遺傳學(xué)的方法檢測(cè)到。

TheBacterioMatch?Two-HybridSystem

大腸桿菌雙雜交系統(tǒng)大腸桿菌雙雜交系統(tǒng)最初是由Karimova等于1998年提出。該檢測(cè)方法是根據(jù)百日咳博代桿菌(Bordetellapertussis)中的腺苷酸環(huán)化酶(CyaA)催化的反應(yīng)為基礎(chǔ)建立的。該腺苷酸環(huán)化酶催化cAMP的合成反應(yīng),其催化結(jié)構(gòu)域可以分成T18和T25兩個(gè)相對(duì)獨(dú)立的功能單位。將所要研究的蛋白質(zhì)分別與T18和T25片斷融合后,再導(dǎo)入大腸桿菌DHP1(cya-)菌株中,如果待檢蛋白質(zhì)間能夠相互作用,那么T18和T25片斷就可借此結(jié)合到一起,催化形成cAMP。合成的cAMP作為一個(gè)信號(hào)分子,可激活作為報(bào)告基因的乳糖或麥芽糖操縱子的表達(dá),產(chǎn)生可以識(shí)別的表型變化。特點(diǎn)和優(yōu)點(diǎn)

雖然酵母雙雜交系統(tǒng)近些年來(lái)得到廣泛的應(yīng)用和不斷的改進(jìn)。但其系統(tǒng)本身仍然還有一些局限。然而大腸桿菌雙雜交系統(tǒng),在某些方面則具有了更大的優(yōu)勢(shì)。第一、研究周期短,操作簡(jiǎn)單。傳統(tǒng)的酵母雙雜交系統(tǒng)需要六天或更久才可得到結(jié)果,而大腸桿菌雙雜交系統(tǒng)只需不到一天的時(shí)間。另外,使用大腸桿菌雙雜交系統(tǒng)時(shí),分離和擴(kuò)增質(zhì)粒都非常簡(jiǎn)單。第二,能夠產(chǎn)生容量更大的文庫(kù)。就目前的方法而言,酵母細(xì)胞的轉(zhuǎn)化效率只能達(dá)到106cfu/μgDNA,而大腸桿菌細(xì)胞可達(dá)到109cfu/μgDNA。第三,更低的假陽(yáng)性率和假陰性率。大腸桿菌的遺傳體系有更小的基因組復(fù)雜性,與高等真核生物相比,又具有更大的進(jìn)化距離。從而,避免了在研究真核生物蛋白相互作用時(shí),由酵母內(nèi)源蛋白所引起的假陽(yáng)性和假陰性現(xiàn)象。此外,一些真核的調(diào)控蛋白有可能對(duì)酵母細(xì)胞產(chǎn)生毒害,而同樣的情況發(fā)生在大腸桿菌中的可能性會(huì)小得多。而且,采用大腸桿菌作為篩選宿主,還能夠避免傳統(tǒng)酵母雙雜交系統(tǒng)對(duì)于核定位的要求。第四,更好的小分子藥物篩選宿主。用酵母雙雜交系統(tǒng)進(jìn)行小分子藥物的篩選時(shí),一個(gè)關(guān)鍵的局限因素在于藥物對(duì)于酵母細(xì)胞膜的可滲透性。然而,大腸桿菌細(xì)胞對(duì)于這些小分子藥物的通透性要好得多。大腸桿菌雙雜交系統(tǒng)的發(fā)展現(xiàn)狀到目前為止,已經(jīng)建立了許多不同體系的大腸桿菌雙雜交系統(tǒng)。根據(jù)其建立原理可分為如下幾類(lèi)。以λ阻遏蛋白為基礎(chǔ)的檢測(cè)系統(tǒng)以協(xié)同抑制為基礎(chǔ)的檢測(cè)系統(tǒng)以形成DNA嚕噗結(jié)構(gòu)為基礎(chǔ)的檢測(cè)系統(tǒng)以酶為基礎(chǔ)的檢測(cè)系統(tǒng)以RNAP為基礎(chǔ)的檢測(cè)系統(tǒng)1以λ阻遏蛋白為基礎(chǔ)的檢測(cè)系統(tǒng)

在λ噬菌體中,阻遏蛋白cI是以同型二聚體的形式發(fā)揮作用的。其每個(gè)亞基由兩個(gè)功能不同的區(qū)組成,N端具有與操縱區(qū)結(jié)合的功能,C端則介導(dǎo)二聚體的形成。而且,一旦移除二聚體形成區(qū)就會(huì)明顯降低DNA結(jié)合區(qū)與操縱區(qū)的結(jié)合力。所以,當(dāng)用外源蛋白代替C端區(qū)域時(shí),只有當(dāng)其發(fā)生相互作用,才能恢復(fù)cI蛋白對(duì)DNA的結(jié)合能力。陽(yáng)性克隆可通過(guò)對(duì)噬菌體感染的免疫力或報(bào)告基因得以檢測(cè)。除了cI蛋白,大腸桿菌來(lái)源的轉(zhuǎn)錄抑制因子LexA,細(xì)菌的阿拉伯糖C基因編碼的AraC等蛋白也已經(jīng)用來(lái)構(gòu)建這種系統(tǒng)。當(dāng)操縱子區(qū)域與啟動(dòng)子區(qū)域有重疊的時(shí)候,阻遏蛋白的結(jié)合就能影響大腸桿菌RNA聚合酶RNAP與啟動(dòng)子的結(jié)合,從而抑制轉(zhuǎn)錄。利用這一原理,將兩個(gè)對(duì)DNA結(jié)合結(jié)構(gòu)域DBD有不同親和力的DNA位點(diǎn)串聯(lián)起來(lái),并使其中對(duì)DBD親和力較低的序列與報(bào)告基因的啟動(dòng)子區(qū)域重疊,這樣當(dāng)與DBD融合表達(dá)的蛋白之間有相互作用發(fā)生,高親和力DBD就可借此穩(wěn)定低親和力DBD與其識(shí)別序列的結(jié)合,從而阻礙RNAP的結(jié)合,抑制報(bào)告基因的表達(dá)。Hays和Hu利用此原理構(gòu)建了一個(gè)檢測(cè)系統(tǒng)。使用的兩個(gè)協(xié)同DNA區(qū)域分別是:對(duì)λ阻遏蛋白有低親和力的λ操縱子序列和對(duì)噬菌體434阻遏蛋白有高親和力的操縱子序列。后者位于前者上游8bp處。λ操縱子序列又與cat-lacZ報(bào)告基因操縱元的-35啟動(dòng)子區(qū)域存在重疊。通過(guò)將Fos和Jun蛋白分別與434阻遏蛋白和λ阻遏蛋白融合的表達(dá),證明了該系統(tǒng)的有效性。2以協(xié)同抑制為基礎(chǔ)的檢測(cè)系統(tǒng)3以形成DNA嚕噗結(jié)構(gòu)為基礎(chǔ)的檢測(cè)系統(tǒng)Kornacker等利用DNA的嚕噗結(jié)構(gòu)可抑制轉(zhuǎn)錄的現(xiàn)象設(shè)計(jì)了該系統(tǒng)。該系統(tǒng)以lacZ作為報(bào)告基因,araBADpromoter為啟動(dòng)子。其上游含有AraC操縱子,可高效結(jié)合AraC。AraC的結(jié)合可激活lacZ的表達(dá)。下游則包含一個(gè)LexA操縱子,其位于lacZ的轉(zhuǎn)錄和翻譯起點(diǎn)之間,由三個(gè)正向重復(fù)的LexA半操縱子區(qū)域組成,可低親合力的結(jié)合LexA。在兩個(gè)上下游操縱子區(qū)域之間還含有一個(gè)整合宿主因子IHF識(shí)別的IHF操縱區(qū)。當(dāng)誘餌蛋白Bait與獵物蛋白Prey分別與AraC和LexA融合后,依靠其相互作用,穩(wěn)定了LexA與其操縱子的結(jié)合,從而引導(dǎo)形成了一個(gè)DNA-loop結(jié)構(gòu),這種結(jié)構(gòu)再由IHF與DNA的結(jié)合而進(jìn)一步加強(qiáng),從而關(guān)閉lacZ基因的表達(dá)。在含有X-gal的培養(yǎng)基上,通過(guò)菌落顏色就可發(fā)現(xiàn)陽(yáng)性克隆。

4以酶為基礎(chǔ)的檢測(cè)系統(tǒng)早在1967年,Ullmann等就將蛋白片段的互補(bǔ)用于基因的結(jié)構(gòu)研究。此后,這種方法用到如蛋白質(zhì)的折疊、酶活性分析和分子進(jìn)化等許多領(lǐng)域。近年來(lái),這一方法逐漸被用到了檢測(cè)蛋白的相互作用。正如前文中所提到的Karimova利用CyaA活性重建而建立的檢測(cè)方法。Michnick等利用鼠二氫葉酸還原酶(mDHFR)的亞基互補(bǔ)特性設(shè)計(jì)了相似的檢測(cè)系統(tǒng)。該法對(duì)菌株是否為falA(編碼DHFR的基因)缺陷型并無(wú)要求,而且可以方便的研究相互作用比較強(qiáng)的蛋白對(duì)。5以RNAP為基礎(chǔ)的檢測(cè)系統(tǒng)

與真核生物的轉(zhuǎn)錄因子有所不同,在原核生物中,許多轉(zhuǎn)錄因子的結(jié)合位點(diǎn)都靠近啟動(dòng)子,并且通過(guò)直接與RNA聚合酶作用激活轉(zhuǎn)錄。1997年,Dove[9]又證明了在DNA結(jié)合蛋白與RNA聚合酶間任意的相互作用都可激活轉(zhuǎn)錄。而且,這種相互作用力越大,對(duì)基因的激活能力也越大。這些發(fā)現(xiàn),導(dǎo)致了一種新的通過(guò)相互作用蛋白直接招募RNA聚合酶激活報(bào)告基因的檢測(cè)方法。在這一方法中,bait質(zhì)粒通常含有λ阻遏蛋白序列,而prey質(zhì)粒常使用RNA聚合酶的α或ω亞基序列。相互作用的蛋白可通過(guò)融合表達(dá)的α或ω亞基招募RNA聚合酶的其他亞基,從而激活報(bào)告基因的表達(dá)。對(duì)于其報(bào)告基因,以前多使用lacZ基因,但Joung等于2000年報(bào)道了一種新的構(gòu)建體系,其中采用了his3和aadA基因作為第一和第二報(bào)告基因??梢越档拖到y(tǒng)的假陽(yáng)性率,而且便于篩選容量更大的文庫(kù)。這一系統(tǒng)是目前大腸桿菌雙雜交系統(tǒng)中最具優(yōu)勢(shì)的系統(tǒng)之一。ComparisonofYeastandE.coliTwo-HybridSystemsConventionalyeasttwo-hybridsystemBacterioMatchTMtwo-hybridsystemGrowthTime6daysormore<1dayandanovernightincubationTransformationefficiency106foryeast109forE.coliDNAmanipulationAdditionaltransformationrequiredintoE.coliDirectcolonyscreeningDNAisolatiomYeastchromosomalDNAco-precipitateswithplasmidUsesasimpleminiprepprocedureGeneralstepsConstructingandcharacterizingabaittransformtheselectstrainofyeastwithbaitandpreyfusedplasmidSelectinganinteractorFirstconfirmationPCR,sequencing,blastSpecificitytestingRetestbyothermeansObtainorconstructcDNAlibraryConstructabaitproteinfusionplasmid,choosereporterplasmidandyeaststrainTransformcDNAlibraryintomatingpartneryeaststrainundernotinducingconditionsFreezeandstoretransformantsdetermineplatingefficiencyofpretransformedlibraryTransformyeast;characterizetranscriptionalactivityofbaitproteintestinglacZandLeureporterCharacterizeexpressionofbaitproteinbywesternand/orrepressionassaySetupmatingInducetheexpressionoflibraryencodedproteinsandselectforinteractingproteinsCharacterizeinsertsbyPCRandrestrictionmapping,chooseindependentclonesTestforspecificitySequencetheinserts,chooseprospectiveisolatesAnalyzetheinteractorsbyothermeansStep1:chooseanappropriatereporterplasmidandyeaststrainstratagenhybridZap:conventionalyeasttwo-hybridsysteminvitrogeneCytoTrapXR:SOSsystemClontech:one-hybridsystemStratagene’sBacterioMatch?Two-HybridSystemStep2:PlasmidconstructionFigure3.Plasmidconstruction.The'bait'and'hunter'fusionproteinsareconstructedinthesamemanner.The'bait'DNAisisolatedandinsertedintoaplasmidadjacenttotheGAL4BDDNA.WhenthisDNAistranscripted,the'bait'proteinwillnowcontaintheGAL4DNA-bindingdomainaswell.The'hunter'fusionproteincontainstheGAL4AD.Figure4.Title.Theyeasttwo-hybridassayusestwoplasmidconstructs:thebaitplasmid,whichistheproteinofinterestfusedtoaGAL4bindingdomain,andthehunterplasmid,whichisthepotentialbindingpartnerfusedtoaGAL4activationdomain.MethodsforPlasmidconstructionStandSubcloningtechniqueHomologousrecombinationNote:Whendecidinghowtoconstructabait,thefollowingpointsareimportant.Theassaydependsontheabilityofthebaittoenterthenucleus,anditrequiresthebaittobeatranscriptionalnonactivator,obvioussequencesthatconferattachmenttomembranesorsequencesthataretranscritionalactivationdomainshouldberemovedfromthechosenprotein.homologousrecombinationStep3:Yeasttransformation-LiAc乙酸鋰轉(zhuǎn)化法:最常用,快捷,105-6轉(zhuǎn)化體/ug-fermentationliquor原生質(zhì)體轉(zhuǎn)化法-Electroporation電轉(zhuǎn)化法-Mating接合轉(zhuǎn)化方法比較轉(zhuǎn)化效率轉(zhuǎn)化數(shù)量花費(fèi)時(shí)間乙酸鋰轉(zhuǎn)化法高適合大規(guī)模的實(shí)驗(yàn)短原生質(zhì)體法較高少對(duì)于大規(guī)模實(shí)驗(yàn)花費(fèi)時(shí)間太多電轉(zhuǎn)化法最高小量DNA轉(zhuǎn)化數(shù)量短Highthroughputyeasttransformations.Mating(接合)在酵母的有性生殖過(guò)程中有2種接合型:a和α。這兩種單倍體之間接合能形成二倍體,但同一接合型間的細(xì)胞不能形成二倍體。將“誘餌”和“獵物”質(zhì)粒分別轉(zhuǎn)化不同接合型的酵母細(xì)胞。過(guò)夜培養(yǎng)混合培養(yǎng)形成的二倍體,在選擇性培養(yǎng)基上篩選發(fā)生相互作用的二倍體。目前用接合方法進(jìn)行大規(guī)模雙雜交的方法可以分成兩種。一種是陣列篩選法,一種是文庫(kù)篩選法。Matrixexperiment(陣列篩選法)用不同接合型的表達(dá)“獵物”和“誘餌”蛋白的酵母株一一接合。用這種方法可以推測(cè)已知的蛋白質(zhì)間的相互作用,例如從最近測(cè)序和注釋的基因組中分析全長(zhǎng)開(kāi)放閱讀框架之間的成對(duì)的相互作用。雖然是一對(duì)一的方法,由于克隆方法和利用小型機(jī)械化的工作站,此方法可用于大規(guī)模的研究。Example:SHIXiaobing,WEIJiamian&SHENYungang,Usingyeasttwo-hybridsystemtodetectinteractionsofATPsynthasesubunitsfromSpinaciaoleraceaFIG.2.Schemefortwo-hybridmatrixexperiments.Detailsaredescribedinthetext.AfterselectionofdiploidsonSc2Leu,2Trp,thepatchesarereplica-platedontothethreedifferentmediaindicated.AnexampleofaSc2Leu2Trp,2Uraplateisshowninwhichoneofthe80pairwisecombinationstestedconferredatwo-hybridpositivephenotype,i.e.,growthonmedialackinguracil.Thefivecontrolsarenotshownhere.Inmatrixexperiments,weusuallyaddthecontrolsonasingleplateforcomparison.文庫(kù)篩選法用一種表達(dá)“誘餌”蛋白的酵母細(xì)胞和一個(gè)表達(dá)復(fù)雜的文庫(kù)“獵物”蛋白的酵母細(xì)胞直接接合。這種高通量的系統(tǒng)可以發(fā)現(xiàn)許多未知的蛋白質(zhì)。然后通過(guò)激活報(bào)道基因來(lái)篩選有相互作用的蛋白質(zhì)。Example:QIBing(齊兵)1QIYipeng(齊義鵬)1MasuoYutsudo2&LIUQingzhen(劉青珍),Isolationandcharacterizationofahumanapoptosisinducinggenewithyeasttwo-hybridsystemStep4:BaitcharacterizationAssayplasmidstotransform

anticipatedresultsreporterLexA-fusionx-galplatesgrowthleu-Glugal/rafGal/rafActivationtestPmw112pBaitwhite/lightbluewhite/lightbluenonegativecontrolpMW112pEG202-RaswhitewhitenoweakpositivecontrolpMW112pEG202-hsRPB7lightbluebluishnostrongpositivecontrolpMW112pSH17-4darkblueblueyesRepressiontestpJK101pBait(white)lighterbluenegativecontrolpJK101-(pGKS3*)whitebluenopositivecontrolpJK101pEG202-RaswhitelighterblueTestbywesternblotExpressiontestuseclonesfrompBaitsinglebandPositivecontrol1activationpEG202-RassinglebandPositivecontrol2assaypSH17-4singlebandDetectionofbaitproteinexpressionWhattodetectTroublesHowtodoAnimportantstepincharacterizationofabaitproteinisthedirectassayofwhetherthebaitisdetectablyexpressedandwhetherthebaitisofthecorrectsize.Inmostcases,bothoftheabovewillbetrue;however,someproteins(especiallywherethefusiondomainis60-80kDorlarger)willeitherbesynthesizedatlowlevels,orbeposttranslationallyclippedbyyeastproteases.Eitherofthesetwooutcomescanleadtoproblemsinlibraryscreens.itisusuallygoodpracticetodemonstratethatthefull-lengthbaitproteinismade.ThiscanbedonebyrunningextractsfromyeastcellsthatharbourthebaitplasmidonanSDSpolyacrylamidegel,immunoblottingwitheitheran

antibodytoLexA

orone

specifictotheproteinfusedtoLexA

,anddetectingafusionproteinoftheexpectedapparentmolecularweight.TroubleshootingandmodificationsofbaitsThebaitactivatestranscription.Thisproblemcanbeadderssedinseveralways.Oneistomakeaseriesoftruncationsoftheprotein,inanattempttoeliminatetheactivationdomain.Ifthebaitactivatestranscriptionverystrongly,thatis,aswellasthepositivecontrol,thisstepwillbenecessary.Ifthebaitactivatesmoderately,thesimplestapproachistore-dothecontrolexperimentsusinglessstringentreporterplasmidsandstrainandtoseewhetheractivationbecomesminor.Alternatively,theproteincanbetruncated,asforastrongactivator.Finally,itispossibletouseanintegratingformofbaitvector,whichwillresultinastablereductionofproteinlevels.Ifthebaitisweakactivator,oneoptionistouselessstringentreporterplasmids;alternatively,theinvestigatormaychoosetoproceedwiththetestedreagents,assumingthatasmallbackgroundoffalsepositivesmaybeidentified.Ingeneral,itisagoodideatousethemostsensitivescreeningconditionspossible;insomecases,useofverystringentinteractionstrainseliminatesdetectionofbiologicallyrelevantinteractions.

Thebaitplasmidproducesaninappropriatelevelorsizeofprotein.Iftheproteinbeingpoorlyexpressedisverylarge,itmaybesimplesttosubdivideintotwoorthreeoverlappingconstructs,eachofwhichcanbetestedindependently.Alternatively,thevectorPjk202incorporatesanuclearlocaliztionsequence,andwillincreasethegeneralconcentrationofthebaitinthenucleus,whereitisrequiredfortheassay.Suchanapproachmaymakethebestofasituationwhereaproteincannotreadilybeexpressedathigherlevels.

Veryfewtransformantscontainingthebaitplasmidexpressthebaitprotein,oryeastexpressingthebaitproteingrownoticeablymorepoorlythancontrolyeastintheabsenceofanyselection.Theseresultswouldsuggestthatthebaitproteinissomewhattoxictotheyeast.Becausetoxicitycancausedifficultiesinperforminglibraryscreening,itmaybedesirabletoreclonetheproteinofinterestintopGilda.Therearethreebasicproblemsthatcanbeidentifiedandpotentiallycorrectedbeforescreening.Table3.possiblemodificationstoenhancebaitperformanceinspecificapplicationbaitproteinresponseStronglyactivatingWeaklyactivatingNottrans-portedtothenucleus,orlowexpressionlevelContinuousexpressionoflexA-fusionistoxictoyeastbaitproteinrequiresunblockedamino-terminalendforfunctionBaitproteinexpressedathighlevels,unstable,orinteractspromiscuouslyPotentialnewproblemTruncate/modifybait+-----Itmaybenecessarytosubdividebaitintotwoorthreeoverlappingconstructs,eachofwhichmustbetestedindependently.Usemorestringentstrain/reporterombination++--+?-+?Useofverystringentinteractionstrainsmayeliminatedetectionofbiologicallyrelevantinteractions.FusetonuclearlocalizationsequencepJK202--+---putLexA-fusedproteinundergal-induciblepromoterpGilda+?--++?+?Cannolongerusegal-dependenceofreporterphenotypetoindicatecDNA-dependentinteraction.FuseLexAtothecarboxylterminusofthebaitpNLexA----+-Generally,lexApoorlytoleratesattachmentoftheamino-terminalfusiondomain;only-60%egratebait,reduceconcentrationpEG202I+?--+?+?+Reducedbaitproteinconcentrationmayleadtoreducedassaysensitivity.+wouldusuallyhelp;+?Mayheop;-willnothelp.Step5:transformingandcharacterizingthelibraryRecommendmatinginthelibraryagainstthebaitofinterestasthemostconvenientstrategy.Themainadvantageofthisapproachisthatiftheinvestigatorwishestousethesamelibrarytoscreenmultiplebaits,onlyasinglelarge-scaletransformationisrequired,followedbyrelativelyeasymatingsteps.Alternatively,withminormodifications,thisprotocolcanbeusedtodirectlytransformthelibraryintoyeastcontainingthebait,whenonlysinglebaitistobescreened.Step6:selectinganinteractor-matingthebaitstrainandpretransformedlibrary-Screenforinteractingproteins:interactorswillbeselectedbyplatingthematedcellsontoselectionmediaplates-Firstconfirmationofpositiveinteractions.-Isolationoflibraryplasmidinsert,andsecondconfirmationofpositiveinteractions:PCR,thePCRproductobtainedinthisstepwillbeusednotonlyforsequencingbutalsoforsecondconfirmationofinteraction.matingthebaitstrainandpretransformedlibraryOncethebaitstrainhasbeenmadeandcharacterizedandthelibrarystrainhasbeentransformedandfrozeninaliquots,thenextstepistomatethetwostrains.Tomatethetwostrains,thebaitstrainisgrowninliquidcultureandthenmixedwithathawedaliquotofthepretransformedlibrarystrain.ThemixtureisthenplatedonYPDandgrownovernight.Duringthistime,individualcellsofthebaitstrainfusewithindividualcellsofthelibrarystraintoformdiploidcells.Themixtrueofdiploidsandunmatedhaploidsisthencollectedandplatedonmediatoselectforinteractors.Inpractice,thediploid/haploidmixtureisgenerallyfrozeninafewaliquotstoallowtiteringandrepeatedplatingsatvariousdilutions.Itisgenerallyagoodideatomatenewbaitstrainswithacontrolstrain.Thecontrolstrainisthesamestrainusedforthelibrary,butcontainingthelibraryvectorwithnocDNAinsert.Matingwiththecontrolstraincanbeperformedatthesametimeasthelibrarymating,andbothmatingscanbetreatedidenticallyinthenextstep,selectinginteractors.ThiscontrolwillprovideaclearestimateofthefrequencyofcDNA-independentfalsepositives,afrequencythatisimportanttoknowwhendecidinghowmanypositivestopickandcharacterize.Growthebaitstrainina30mlGlu/CM-Ura-Hismasterplatewithshakingat30Ctolate-logphase(OD600=1.0-2.0)Collectthecells:1000g,5min.ResuspendcellsinsterileH2Oandtransfertoasterile1.5-mlmicrofugetube.Thawanaliquotofthepretransformedlibrarystrainatroomtemperature.Mix200ulofthebaitstrainwith-108cellsofthepretransformedlibrarystrain.Centrifugethemixtureofcells,1000g5minanddiscardthesupernatant.Resuspendthecellpelletin200ulofYPDmedium.Plateonasingle100mm-diameterYPDplateandincubatefor12-15hat30c.Add1-2mlofsterileH2OtothesurfaceoftheYPDplateandresuspendthecellswhileshakingwithsterileglassbeads.Transfertosterilecollectiontubesandvortexgentlyfor2trifugation,1000g,5min,resuspendin1volumeofsterileglycerolsolution.Distributeinto200-ulaliquotsandfreezeat-80c.matingthebaitstrainandpretransformedlibraryGeneralstepsScreenforinteractingproteinsThawonealquotofthematedyeast(orcellscontainingpBaitthatweretransformeddirectlywiththelibraryDNA)withthelibraryDNA)anddilute1:100withgal-Raff/CM-Rra,-His,-Trpdropoutmedium.Incubatewithshaking4-6hat30ctoinducethegal1promoteronthelibrary.Plate-1X106cellson100mmgal-Raff/CM-Ura,-His,-Trp–LEUdropoutplates.Incubatetheplates5daysat30c.Goodcandiatesforpositiveinteractorswillgenerllyproducecoloniesoverthistimeperiod,dependingontheindividualbaitused.Thecommonappearanceofcoloniesshouldbeat2-4daysafterplating.GeneralstepsInsomecases,nopositivesareobtainedforagivenbait.why?Thelibrarysourcewasinappropriateorthataninsufficientnumberofcolonieswerescreened.Thebaitdoesnot,infact,interactwithanysinglepartnerproteinwithsufficientaffinitytobedetected.Thebaitisnotinnativeformation,eitherbecauseofadisfavoredtruncationorbecauseofstericinteractionswiththefusiondomain,orthatthebaitisnotinteractingwellwiththelexAoperatorbecausesomemoietyonthefusionproteinisrequesteringtheprotein.Sometimesitispossibletomodifybaitandscreeningconditionstoobtaininteractors;however,somebaitssimplydonotappeartogenerateinteractors.FirstconfirmationofpositiveinteractionsThefollowingstepstestforgalactose-dependenttranscriptionalactivationofboththelexAop-Leu2andlexAop-lacZreporters.Simultaneousactivationofbothreportersinagalactose-specificmannergenerallyindicatesthetranscriptionalphenotypeisattributabletoexpressionoflibrary-encodedproteins,ratherthanderivedfrommutationoftheyeast.step7:specificitytestingThisstepistodeterminewhetherisolatedcDNAsarerepeatableandspecifictothepBaitofinterest,andtoexcludelibrary-encodedcDNAsthatinteractwithlexA(insteadofthebait),stickyproteinsthatinteractwiththepBaitinanonspecificmanner,andclonesisolatedbecauseofmutationsintheinitialSKY48strainthatrendergrowthandtranscr

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