基因突變庫的篩選方法課件

基因直接進(jìn)化研究進(jìn)展

——DNAShuffling技術(shù)四川農(nóng)業(yè)大學(xué)二○○三年九月吳琦生物的自然進(jìn)化進(jìn)化過程：突變→自然選擇→遺傳后代進(jìn)化結(jié)果：

基因多樣性：為完成同一功能所表現(xiàn)出的多個基因或同一個基因(同源性)

代謝途徑的多樣性：同樣產(chǎn)物，多條途徑

代謝產(chǎn)物的多樣性：同一底物，不同產(chǎn)物

生物多樣性：整個生態(tài)系統(tǒng)中的生物人工獲取新基因的方法常規(guī)的基因工程方法生物功能蛋白質(zhì)基因新基因的理性設(shè)計(jì)和人工合成

根據(jù)已有基因的序列和功能進(jìn)行設(shè)計(jì)基因的直接進(jìn)化（directedevolution)

可使已有基因獲得新的特性可獲得自然界中不存在的基因可解決許多新的理論和應(yīng)用問題TLPs的失活曲線TLP-ste突變蛋白的三維結(jié)構(gòu)酶拓?fù)浣Y(jié)構(gòu)的變化基因直接進(jìn)化的步驟突變

基因突變庫的建立篩選

基因突變庫的活體或離體篩選基因復(fù)制與遺傳同序法（consensusapproach）對一組同源蛋白質(zhì)的氨基酸序列進(jìn)行比較，以一定的標(biāo)準(zhǔn)程序計(jì)算出各氨基酸序列的共有序列；人工合成同序基因后重組表達(dá)。MartinLehmann等（2000～2002）把這種方法應(yīng)用在真菌植酸酶家族設(shè)計(jì)合成了同序植酸酶基因，并表達(dá)出具熱穩(wěn)定性的同序植酸酶。同序植酸酶-1的最適溫度為71℃，而親代植酸酶的最適溫度為45-55℃，增加了16-26℃,同序植酸酶-1Tm為78℃，比親代植酸酶增加了15-22℃，而催化性質(zhì)與大多數(shù)親本植酸酶相似。DNAShuffling:外顯子、單基因和基因家族的重組裝隨機(jī)引物延伸法交錯延伸法隨機(jī)片段活體突變:

線狀基因和隨機(jī)片段共轉(zhuǎn)化酵母細(xì)胞基因突變庫的篩選方法ScreeningtheLibraryandSelectingtheRightClone

平板分析使抗生素失效的酶類(如頭孢菌素酶，b

-乳糖酶）提高耐熱性易于觀察的菌落表型（如菌落顏色等）營養(yǎng)缺陷型的輔助篩選噬菌體展示、細(xì)胞表面展示根據(jù)所需要的特性，對經(jīng)Shuffling后的DNA文庫在噬菌體或細(xì)菌細(xì)胞表面（細(xì)菌、纖毛蟲細(xì)胞表面）的表現(xiàn)特征進(jìn)行篩選，獲得提高目的底物親和力的突變子。減少篩選工作量突變文庫→大的突變子庫→小的突變子庫→單克隆噬菌體展示篩選模式WhathappensafterDNAshufflingGenerationofalargelibraryofnovelgenes(chimeras)Selectionforimproved/desiredbio-functions

crossovers,deletions,insertions,inversions,

pointmutationsDarwinianEvolution

NaturalselectionofexistingmutationsDirectedEvolution

TargetedselectionofcreatedmutationsWhatisDNAshuffling?DNAShuffling的內(nèi)涵DNAShuffling：指DNA分子的體外重組，是基因在分子水平上進(jìn)行有性重組(SexualRecombination)。通過改變單個基因(或基因家族，genefamily)原有的核苷酸序列，創(chuàng)造新基因，并賦予表達(dá)產(chǎn)物以新功能。DNAShuffling的內(nèi)涵HowDNAshufflingworks-1HowDNAshufflingisdoneinthetube

Randomfragmentationofapoolofrelatedgenes;Self-primingpolymerasereactionandtemplateswitching(causingcrossovers);PCRamplificationwithprimersofreassembledproducts

HowDNAshufflingworks-2Similarmutantsgeneratedbyerror-pronePCR,randomandsite-directedmutagenesis...........SinglegeneshufflinglibraryofpointmutantsFamilygeneshufflinglibraryofchimerasGeneratingchimeraswithcrossoversoflargeblocksofsequencesDNA重組裝的過程隨機(jī)引物PCR和重組裝磷脂酶熱穩(wěn)定－催化活性的分子進(jìn)化（JaeKwangSong等，2000）DNAShuffling技術(shù)的應(yīng)用枯草桿菌蛋白酶E熱穩(wěn)定性的分子進(jìn)化（HuiminZhao等，1999）進(jìn)化后的枯草桿菌蛋白酶E正面反面耐熱p-硝基苯酯酶的分子進(jìn)化(LoriGiver等，1998）β-葡糖苷酶耐熱性的提高(Mary′aJesu′sArrizubieta等，2000）EvolutionofacytokineusingDNAfamilyshuffling

Chia-ChunJ.Chang…….PhillipPatten

NatureBiotechnologyVol.17,August,1999Aimsofthework:DemonstrationofusefulnessoftheDNAshufflingtoolforrapidlyevolvinghumanainterferontowardsimprovingantiviralpropertyInterferons(IFNa,IFNb,IFNg)Reasonsfortheirlimiteduseasdrugs: IFNasareproducedbymonocyte,macrophagesandothercellsuponstimulationbyvirus.

IFNsareabletoprotectcellsfromviralinfectionbyreactingwithIFNreceptorsonthecellwhichtriggersaseriesofsignaltransductionevents,henceimmuneresponse.

IFNssuppressanexaggeratedgrowthpotentialofcancercells.

IFNscanbeusedasdrugsinthetreatmentofviralinfection.OnlyonenaturalIFN-a,Hu-IFN-a2,inclinicalstudies.MostactiveengineeredIFN-a,alfacon-1,isaconsensusof13wildtypeHu-IFN-agenesthatisusedinhepatitisCtherapy.Dose-limitingtoxicity,Receptorcross-reactivity,Shortserumhalf-livesMethodsandMaterials-1DNAShuffling

Constructionof1stroundlibrary:8clonedHu-IFNgeneswereshuffledandassembledinsertclonedinthephagemiddisplayvector

Constructionof2ndround5libraries:

IFN-CH1.1xIFN-CH1.2;

IFN-CH1.1xIFN-CH1.

3;

IFN-CH1.

1xIFN-CH1.4;

IFN-CH1.2xIFN-CH1.

3;

IFN-CH1.

1xIFN-CH1.

2xIFN-CH1.

3xIFN-CH1.

4(matingof4genes).Pair-wisematingsPhagemiddisplayofIFNHigh-throughputphagemid

preps(Q-BOTcolonypicker)Antiviralassays:CytopathiceffectreductionassayonmouseL929cells,challengingvirus-EMCV,96wellplateformat,neutralred,cellfixing,colourextracting,specOD540vsIFNaconcentration.phagemidsE.coliExpressioninducedphagemidsaggregatedPEGprecipCsClbandedLibraryintro-intoE.coliThousandsofsingleclonespickedbyrobot,placedin16x96wellplatesExpressioninduced,phagemidsaggregatedpackaged,released16pools96assayedActivepoolbrokeninto8poolsof12All8poolsassayedwith3havingactivityAllthe36clonesinthe3poolswereprepared,purified&assayedDeconvolutionoflibraries-screeningstrategyShuffledproductsSo16+8+36=60assayswerecarriedoutinsteadof16x96=1536MethodsandMaterials-2CHO(ChineseHamsterOvary)expressionandpurification

(Histag)ofchimericIFN-as.

High-throughputproliferationassays(inhibitionofproliferation)inmouseL929cells:

Standard[3H]thymidineincorporationmethods--IFN-asamplesweremixedwithL929cellsin96wellplates,incubatedand[H3]thymidineaddedtoeachwell.PlateswereharvestedonHarvester-96andthymidineincorporationwascountedonabcounter

HumanDaudicellproliferationassays:

4mostactivechimerasandHu-IFN-a2awereexpressed,purifiedandassayedforanti-proliferationactivityonhumanDaudicellsMethodsandMaterials-3Results-1:structureofshuffledIFNasB:Thesequenceofoneofthecycle2chimeras,IFN-CH2.2,isalignedwiththemostpotenthumanandmouseIFN-as.TheIFN-aresiduesthatputativelycontacttheIFN-areceptorareboxed.ResiduesinHu-IFN-a1thathavebeenshownbysite-directedmutagenesistocontributetoactivityonmousecellsareshaded.Results-2:ActivityofparentalandshuffledIFN-asinmurinecellsResults-AntiviralactivityrankingAntiviralactivityResults-PotencyofIFN-asLog10[IFN-a](mg/ml)OD540nm(cellviability)

Results-continuedStructuralmodellingofIFN-CH2.2basedontheNRMstructureofHu-IFN-a2a.TheproteinbackboneiscolouredtoindicatethenativeHu-IFN-asegments(5Hu-IFN-asfromwhichitisderived）.ThesidechainsofputativemurineIFN-areceptorcontactingresiduesLys121andArg125areshownConclusions1.Unlikesite-directedmutagenesisandotherproteinengineeringmethodswhichproducemainlypointmutations,DNAfamilyshufflingcangeneraterecombinantswithblocksofsequencesfrom