基因突变库的筛选方法课件

基因直接进化研究进展

——DNAShuffling技术四川农业大学二○○三年九月吴琦生物的自然进化进化过程：突变→自然选择→遗传后代进化结果：

基因多样性：为完成同一功能所表现出的多个基因或同一个基因(同源性)

代谢途径的多样性：同样产物，多条途径

代谢产物的多样性：同一底物，不同产物

生物多样性：整个生态系统中的生物人工获取新基因的方法常规的基因工程方法生物功能蛋白质基因新基因的理性设计和人工合成

根据已有基因的序列和功能进行设计基因的直接进化（directedevolution)

可使已有基因获得新的特性可获得自然界中不存在的基因可解决许多新的理论和应用问题TLPs的失活曲线TLP-ste突变蛋白的三维结构酶拓扑结构的变化基因直接进化的步骤突变

基因突变库的建立筛选

基因突变库的活体或离体筛选基因复制与遗传同序法（consensusapproach）对一组同源蛋白质的氨基酸序列进行比较，以一定的标准程序计算出各氨基酸序列的共有序列；人工合成同序基因后重组表达。MartinLehmann等（2000～2002）把这种方法应用在真菌植酸酶家族设计合成了同序植酸酶基因，并表达出具热稳定性的同序植酸酶。同序植酸酶-1的最适温度为71℃，而亲代植酸酶的最适温度为45-55℃，增加了16-26℃,同序植酸酶-1Tm为78℃，比亲代植酸酶增加了15-22℃，而催化性质与大多数亲本植酸酶相似。DNAShuffling:外显子、单基因和基因家族的重组装随机引物延伸法交错延伸法随机片段活体突变:

线状基因和随机片段共转化酵母细胞基因突变库的筛选方法ScreeningtheLibraryandSelectingtheRightClone

平板分析使抗生素失效的酶类(如头孢菌素酶，b

-乳糖酶）提高耐热性易于观察的菌落表型（如菌落颜色等）营养缺陷型的辅助筛选噬菌体展示、细胞表面展示根据所需要的特性，对经Shuffling后的DNA文库在噬菌体或细菌细胞表面（细菌、纤毛虫细胞表面）的表现特征进行筛选，获得提高目的底物亲和力的突变子。减少筛选工作量突变文库→大的突变子库→小的突变子库→单克隆噬菌体展示筛选模式WhathappensafterDNAshufflingGenerationofalargelibraryofnovelgenes(chimeras)Selectionforimproved/desiredbio-functions

crossovers,deletions,insertions,inversions,

pointmutationsDarwinianEvolution

NaturalselectionofexistingmutationsDirectedEvolution

TargetedselectionofcreatedmutationsWhatisDNAshuffling?DNAShuffling的内涵DNAShuffling：指DNA分子的体外重组，是基因在分子水平上进行有性重组(SexualRecombination)。通过改变单个基因(或基因家族，genefamily)原有的核苷酸序列，创造新基因，并赋予表达产物以新功能。DNAShuffling的内涵HowDNAshufflingworks-1HowDNAshufflingisdoneinthetube

Randomfragmentationofapoolofrelatedgenes;Self-primingpolymerasereactionandtemplateswitching(causingcrossovers);PCRamplificationwithprimersofreassembledproducts

HowDNAshufflingworks-2Similarmutantsgeneratedbyerror-pronePCR,randomandsite-directedmutagenesis...........SinglegeneshufflinglibraryofpointmutantsFamilygeneshufflinglibraryofchimerasGeneratingchimeraswithcrossoversoflargeblocksofsequencesDNA重组装的过程随机引物PCR和重组装磷脂酶热稳定－催化活性的分子进化（JaeKwangSong等，2000）DNAShuffling技术的应用枯草杆菌蛋白酶E热稳定性的分子进化（HuiminZhao等，1999）进化后的枯草杆菌蛋白酶E正面反面耐热p-硝基苯酯酶的分子进化(LoriGiver等，1998）β-葡糖苷酶耐热性的提高(Marý´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