定点突变与重组蛋白表达.ppt

定点突变与重组蛋白表达,DNArecombination,将不同来源的DNA片段通过末端共价连接形成重新组合的DNA分子的过程。,Genecloning,在体外对DNA分子按照既定的目的分离，剪切和重新连接，构成重组的DNA分子，并在特定的受体细胞中，与载体一起得到复制和表达，从而获得该分子的大量拷贝。,AspecificDNAfragmentisisolatedfromlargegenomicDNAandligatedtoavectorDNA(relativelysmall)byusingrestrictionenzymesandligases;Therecombinant(hybrid)DNAmoleculeisthendeliveredintoahostcell(oftenbeingE.colicells)andamplifiedaccompanyingcellpropagation.,第一节工具酶及载体限制性核酸内切酶（restrictionendonucleases）Restrictionenzymes(通常是二聚体)能识别并且切割特定的具有回文结构的序列(HamiltonSmith,1970);能识别双链DNA分子内部的特异位点并且裂解磷酸二酯健。,根据酶结构，识别切割序列上的特异性，催化条件及修饰活性等分为：I型多亚基蛋白复合物，兼具限制酶甲基化酶活性，需Mg,ATP,SAMII型识别切割具极强特异性。由两个亚基组成，需MgIII型相当少，有两个亚基，切割缺乏特异性,SometypeIIrestrictionenzymescleavebothDNAstrandsatthesamesiteleavingtwobluntendswithoutunpairedbasepairs,OthertypeIIrestrictionenzymesmakestaggeredcutsonthetwoDNAstrandsleavingtwocohesiveends(withprotrudingsinglestrand)whichcanformbasepairwitheachotherorwithanyotherDNAfragmentswithcomplementarycohesiveends;PstI5-CTGCAG-35-CTGCAG33-GACGTC-53-GACGTC5,限制酶反应的影响因素1.温度一般37，也有例外SmaI为25。2.缓冲液不同的酶对离子强度，pH有不同要求3.时间通常11.5hr4.反应体积加酶体积一般为总反应体积1105.DNA的纯度和结构,二.连接酶和末端转移酶TwodifferentDNAmoleculescutwiththesameorcompatiblerestrictionenzymescanbeligatedintoonerecombinantDNAmoleculebyusingDNAligases;T4-DNAligaseThefirstrecombinantDNAmoleculewasmadewithlphageandSV40DNAmoleculesbyusingterminaltransferasetomakestickyendsontwodifferentDNAmolecules(PaulBerg,1972).,三.载体（vector)质粒染色体外能自主复制的共价双链闭合环状DNA分子，广泛存在于原核细胞内。质粒DNA的大小：1200kb小的能编码23种中等大小的蛋白质，其分子量约为106dal，而大的质粒分子量则可高达108dal,分子量相对较小具有可供选择的遗传标志有尽可能多的限制酶切点具复制起点，能自主复制Plasmidvectorsmustatleastcontain:areplicationorigin,anantibioticresistancegene(functionasaselectablemarker),andarestrictionsitewhereforeignDNAcanbeinserted(upto15kbcanbecloned);,2.噬菌体BacteriophagelcloningvectorscanbeusedtoclonelargerDNAfragments,wheretherecombinantDNAispackedintophageparticlesinvitroandthenbedeliveredintoE.colicellswithhighefficiency(upto23kbcanbecloned);插入型载体,容量较小，gt11具有一个限制酶位点可供外源DNA片段插入；容量为012kb。置换型载体具有成对的限制酶位点，在这两个位点之间的DNA区段可以被插入的外源DNA片段所取代；容量为923kb,UsingbacteriophagelDNAascloningvectorinE.colicells.,OnlyrecombinantofappropriatesizeandhavingthetwoessentialphageDNAfragmentswillbepackedintophageparticles,EnterE.colicells,ADVANTAGESOFTHElSYSTEM1.Largeinsertspossible(upto20kb).2.Stablepropagation(C.F.plasmidswherelargeinsertsmaygetdeleted).3.Efficiententryintocellusinginvitropackaging.4.Multiplecopiesofinsertandstrongpromoters.DISADVANTAGESOFTHElSYSTEM1.Plaquesareproducednotcolonies.UsefulforcloningDNAbutnouseforgeneexpression,ductionofgeneproductsinbiotechnology.2.20kbisstillnotlargeenoughformanyeukaryoticgenes.Forlargerinsertscosmidsoreukaryoticvectorscanbeused.,3.黏性质粒含有l噬菌体的黏性末端有质粒的抗药性标记特点具多个MCS本身分子量小，可容纳45kbDNA体外包装主要是重组体，利于筛选CosmidvectorscontainfeaturesofbothplasmidandphageacceptingevenlargerDNAfragments(upto45kb).,Artificialconstructions.ConsistofthecosendsofphagewithplasmidDNA.InvitropackagingisusedtointroducetheDNAintoahostcell.AnyDNAbetween2cossiteswillbepackagedinthephagehead.PhageinjectstheDNAwhichthenbehaveslikeaplasmidonceinsidethehostcell.Butsuchphagesarenotvirulentanddonotcauselysisorproduceplaques.Coloniesareproduced.Thereforegeneexpressionandproductionispossible.Insertsupto40kbcanbeinserted.Selectioncanbebyaddingampicillinresistancegenetoinsert-positiveselectiononampicillinplates.However,cosmidscanbeunstable.,ThecosmidDNApropagatesasaplasmidinE.colicells,Largeoneswillbepackedintophageparticles,SmalloneswillbetransformedintoE.colicells.,Forpackagingintolphageparticles,4.表达载体（expressingvector)用来在宿主细胞中表达外源基因的载体，除具有克隆载体的性质外，还带有转录和翻译所必需的DNA序列。原核细胞表达载体，E.coli真核细胞表达载体yeast,mammaliancell,制备目的基因构建重组体将重组体转入宿主细胞重组体的筛选和鉴定重组体的扩增和表达,基因克隆的基本程序,第二节基因重组,一.目的基因的制备,1.Genesareisolatedfromagenomiclibrary,基因文库是收集来源于某种有机体基因组DNA的所有片断(通过某种限制性内切酶切割后,e.g.,Sau3A)，分别将这些片断连接到克隆载体中(质粒,l噬菌体,cosmid,orYAC);,基因文库要包含所有的编码和非编码DNA序列.,侵染E.coli细胞获得嗜菌斑,由l载体构建基因文库,2.GenesareisolatedfromacDNAlibrary,cDNA文库是收集所有来源于某种有机体、某种细胞或组织mRNAs的DNA分子片断(用反转录酶从互补DNA转化而来,亦称cDNA,)而建立起来的。AcDNAlibraryisa(complete)collectionofDNAfragmentsderivedfromthemRNAs(convertedtotheircomplementaryDNA,orcDNA,usingreversetranscriptase)ofaparticularorganism,certaincellsortissuesofanorganism.,AcDNAlibraryisgeneratedfromamRNA“library”fromacertainsource.,InsertintoacloningvectortomakeacDNAlibrary.,3.SpecificDNAsequencescanbeamplifiedusingpolymerasechainreaction(PCR),ApairofoligonucleotideprimerscomplementarytotheflankingregionsofthetargetDNAhavetobesynthesizedRepeated(usuallyabout30cycles)chainseparationandDNAsynthesis(usingthethermostableTaqDNApolymerase)willamplifythetargetDNAmillionsoffold;ThePCRtechnique,inventedbyKaryMullisin1984,causedarevolutionforDNAdetectionandmanipulation.,4.Genearesynthesizedbychemicalmethod,二.目的基因与载体重组,黏性末端连接平端连接人工头连接同聚物加尾连接,三.重组DNA导入宿主细胞,Transformation转化，质粒及重组体导入细菌Transfection转染，病毒及重组体导入宿主细胞Transduction转导，噬菌体及重组体导入宿主细胞,1.氯化钙法特点：转化率低,2.电穿孔法特点：操作简单，转化率高；穿孔条件难掌握，细胞杀伤率高,3.脂质体转染法特点：转化率最高，但脂质体昂贵,4.显微注射法特点：仪器昂贵，操作需熟练,四.重组体的筛选和鉴定,1.遗传学方法插入失活,蓝白筛选,2.限制性内切酶图谱鉴定3.核酸杂交法原位杂交4.PCR筛选法5.免疫化学筛选法,第三节定点突变,寡核苷酸介导的基因突变盒式突变或片段取代突变PCR定点突变,随机突变,基因突变,位点特异性突变,定点突变(site-directedmutagenesis)在体外特异性改变某个碱基的技术,OligonucleotidemismatchmutagenesiscancreateadesiredpointmutationatauniquepredeterminedsitewithinaclonedDNAmolecule.,site-directedmutagenesistheresearcherscanstudyindetailhowproteinsfunctionandhowtheyinteractwithotherbiologicalmolecules.Site-directedmutagenesiscanbeused,forexample,tosystematicallychangeaminoacidsinenzymes,inordertobetterunderstandthefunctionoftheseimportantbiocatalysts.Theresearcherscanalsoanalysehowaproteinisfoldedintoitsbiologicallyactivethree-dimensionalstructure.Themethodcanalsobeusedtostudythecomplexcellularregulationofthegenesandtoincreaseourunderstandingofthemechanismbehindgeneticandinfectiousdiseases,includingcancer.,1.影响的各种因素,一.寡核苷酸介导的位点特异性突变,1）DNA模板的制备,2）引物的设计和选择引物的特性引物的设计,3）突变引物与模板DNA的退火和引物延伸条件,4）关于DNA聚合酶的选择,5）受体细胞对突变体产率的影响,2.几种寡核苷酸介导的基因突变方法,Kunkel突变法,1)将待突变的基因克隆到质粒中，导入具有ung-dut-双缺陷的E.coli中.dut-(缺少dUTPase，导致细胞内dUTP水平上升)细菌聚集dUTP，并在DNA复制时，部分取代dTTP进入DNA新生链。ung-(缺少尿嘧啶脱糖苷酶)细菌不能去除掺入的dUTP，最终的结果是，转入到细菌中的质粒DNA中，大约由1的T被U所取代。,Figure1.dsDNAplasmidistransformedintoung-bacteriawhichconvertstheTstoUs.Inthisexample,theGCbasepairwillbetargetedformutagenesis.ThisU-containingplasmidisconvertedtosingle-strandedDNA.,2)含有U的目标DNA与含有突变的寡核甘酸片断孵育，该寡核甘酸片断除了需要的突变点外，都与目标DNA配对。这种突变可以是一个或几个核甘酸的插入、删除和碱基替换。然后该混合物与Klenow,dNTPs孵育，接着加入连接酶和ATP产生双链的质粒，其中一条链含有U，而新合成的链只含有T。需要注意的是突变的核甘酸与旧的模板链是不配对的。,Figure2.TargetplasmidisusedasatemplatetoproduceasecondstrandofDNAthatcontainsthedesiredmutationandonlyTs,3)最后，杂交的双链DNA被转入到正常的细菌中，其尿嘧啶糖基化酶除去DNA链上的尿嘧啶碱基，原来的模板链被降解，只有突变链因不含U，被保留下来。再以此突变链为模板合成出来的新链都含有突变碱基。最终所有的质粒都含有新的突变碱基。,Figure3.Hybridplasmidistransformedintowild-typebacteriathatconverttheoriginalU-containingstrandofDNAintoastrandofT-containingDNAthatiscomplementofthemutatedsequence(ingreen).TheoriginalGCbasepairhasbeenconvertedtoanewTAbasepair.,抗生素抗性回复突变法此体系利用pALTER-1噬菌粒为突变载体。Thesystemsuseantibioticselectionasameanstoobtainahighfrequencyofmutants.特点：1.有MCS2.含Amps,Tetr3.利用Helperphage,previous,PromegaCorporation2800WoodsHollowRoadMadison,WIUSAContactUs608-274-4330,pALTER-1载体含氨苄青霉素和四环素两个抗性基因,但是氨苄青霉素的抗性基因是没有活性的。氨苄修复寡核甘酸可以在突变反应中恢复突变链的氨苄的抗性。ThepALTER-1Vectorcontainsgenesforampicillinandtetracyclineresistance,buttheampicillinresistancegenehasbeeninactivated.TheAmpicillinRepairOligonucleotiderestoresampicillinresistancetothemutantstrandduringthemutagenesisreaction.,适合的寡核甘酸片断可以同时用在突变反应中，去除一个抗生素活性的同时而修复另一种抗生素活性。用这种方法随后的突变和选择可以在同一质粒上进行，而不需要亚克隆。Theappropriateoligonucleotidescanbeusedsimultaneouslyinthemutagenesisreactiontoinactivateoneresistancegenewhilerepairingtheother.Inthisway,subsequentroundsofmutagenesisandselectioncanbeperformedonthesameplasmidwithoutsubcloning.,特点：多克隆位点，用以对目标基因的克隆。含有四环素(Tetr)和氨苄青霉素(Amps)的抗性基因,在突变中用氨苄青霉素抗性修复寡核甘酸使AmpsAmpr,用于对阳性克隆的筛选。利用辅助噬菌体超感染，制备含目标基因的单链DNA作为突变模板DNA。为提高突变效率，受体菌应是点错配修复缺失的菌株。,基于去除特定限制酶切位点的突变法在突变载体DNA上除了有克隆外源基因的克隆位点外，还有一独特的限制酶切位点。,该,该位点也不存在目标基因上,突变时用两个引物：一是突变引物；二是独特限制酶切位点去除的选择性引物。,优点：不需制备单链DNA;不需辅助噬菌体；不需进行亚克隆。,优点：方便、快捷、不受限制性内切酶识别位点的限制,二.利用PCR产生定点突变,利用PCR法在基因5末端区或3末端区产生突变2.重叠延伸(overlapextension)和基因突变,加到PCR引物5端的序列掺入到PCR产物的末端。一个PCR扩增片段可与另一个PCR扩增片段上的序列相重叠，其后的反应中，这段重叠序列可以作为引物被DNA聚合酶所延伸，由此产生一个新的重组分子。,非生产链,生产链,几点补充：重叠延伸反应中非生产链的命运？重叠延伸反应进行基因剪接，其不同部位在反应中执行不同功能引导区(primingregion):寡核苷酸3端重叠区(overlapregion):寡核苷酸5端插入区：重叠区和引导区之间,盒式突变（cassettemutagenesis),又称片段取代法（DNAfragmentreplacement)关键1.目标基因序列中有适当限制酶切位点2.如何得到各种合适的用以取代目标基因中特定DNA片段的突变DNA片段,三.区域性定向突变,四.随机突变（randommutagenesis),当缺乏目的基因序列的详尽资料时，定点突变就受到限制，此时可利用随机突变。,待突变基因克隆到载体上，其下游紧接两个限制酶切位点。,前一酶切位点是5突出，后一酶切位点是3突出,ExoIII催化线性双链DNA或有缺口环状DNA自3-OH端逐一释放5单核甘酸。,适当时机终止酶切反应,缺口填补，类似物掺入DNA链的一处或多处,S1核酸酶处理单链末端，形成平末端,50基因携带有错配的碱基，导致位点变异,第四节重组蛋白质的表达,一.目标蛋白在E.coli中的表达,在E.coli中表达蛋白是非常简单,快速和便宜的。有很多商品化和非商品化的载体，在N-末端或C-末端有标签（tag),并有很多菌株供使用者选择。TheexpressionofproteinsinE.coliistheeasiest,quickestandcheapestmethod.Therearemanycommercialandnon-commercialexpressionvectorsavailablewithdifferentN-andC-terminaltagsandmanydifferentstrainswhichareoptimizedforspecialapplications.,优点1.遗传学和生理学背景清楚2.容易培养，特别是高密度发酵3.外源基因常可以高效表达,不足1.不能进行典型真核细胞所具有的复杂的翻译后修饰2.限制二硫键的形成，多亚基复合体的组装3.外源基因易形成不溶性的包涵体,表达载体（expressingvector)用来在宿主细胞中表达外源基因的载体，除具有克隆载体的性质外，还带有转录和翻译所必需的DNA序列。,原核细胞表达载体，E.coli真核细胞表达载体，yeast,mammaliancell,RecombinantproteinscanbeproducedinE.colicellsusingspecificexpressionvectors.,BasicelementsformakinganE.coliexpressionvector,表达载体的一般特点复制起始点选择性基因强的，可诱导的启动子强的转录终止序列核糖体结合位点合适的多克隆位点,2.与外源基因有效表达的相关因素1）有效的转录起始2）有效地翻译3）蛋白质水解作用4）蛋白质的外泌,3.蛋白质表达的方法,Atypicalexpressionexperimentconsistsofthefollowingstep:Pickingofasinglecolonyfromafreshlystreakedplateoftheexpressionhostcontainingtherecombinantvector.Growingofastarterculture.Inoculate(接种)withthepickedcolonyupto50mlofrichmedium(suchasLBor2xYT)containingtheappropriateantibiotic.Whenalargerstartercultureisrequired,inoculate4mlofrichmediawiththesinglecolony;growfor4-8hoursat37C;andusethistoinoculatethestarterculture.,InoculationofthemaincultureandincubationuntilOD600reaches0.4-1.TheoptimalODvaluedependsontheculturemethodandthemedium.ForflaskculturesusingLB-mediumanOD600of0.6isrecommended.Toincreasethegrowthrate,wecarryouttheculturesat37CuntiltheODforinductionisreached.Thentheculturesarecooledtotheinductiontemperatureinice-water.Inductionofproteinexpression.Proteinexpressionisinducedbytheadditionoftheproperinducerorbychangingthegrowthconditions.Fromthispointonthecellswillusemostoftheirresourcesfortheproductionofthetargetproteinandwillnotgrowmuchfurther.,Forthemostusedpromotersinductionconditionsarelistedbelow.Promoterinductiontypicalconditionrangetrc(hybrid)additionofIPTG0.2mM0.05-2.0mMaraBADadditionofl-arabinose0.2%0.002-0.4%PLliftingthetemperaturefrom37to42CT7-lacoperatoradditionofIPTG0.2mM0.05-2.0mM,Afterinductiontheculturesareincubatedfrom3hourstoovernightdependingontheinductiontemperature.Incubationtemperatureincubationtime15Covernight20Covernight25Covernight30C5-6h37C3-4hHarvestingofthecellpelletbycentrifugation(20minat6000g).Cellpelletsarestoredat-20C.,4.改善表达水平及溶解性的方法1）如何改善表达水平诱导条件外源基因的编码序列用蛋白酶缺失的宿主菌,2）如何改善外源蛋白的溶解性,Inmanycasestheexpressedproteinisinsolubleandaccumulatesinso-calledinclusionbodies(包含体).Thisisespeciallytrueunderconditionsofhighlevelexpression.Severalstrategiesareavailabletoimprovethesolubilityoftheexpressedprotein.,1.Reducingtherateofproteinsynthesis.Thiscanbedoneby:loweringthegrowthtemperature.Thisdecreasestherateofproteinsynthesisandusuallymoresolubleproteinisobtained.usingaweakerpromoter(e.g.trcinsteadofT7).usingalowercopynumberplasmid.loweringtheinducerconcentration.,2.Changingthegrowthmedium.additionofprostethicgroupsorco-factorswhichareessentialforproperfoldingorforproteinstability.additionofbuffertocontrolpHfluctuationinthemediumduringgrowth.additionof1%glucosetorepressinductionofthelacpromoterbylactose,whichispresentinmostrichmedia(suchasLB,2xYT).additionofpolyols(e.g.sorbitol)andsucrose.Theincreaseinosmoticpressure(渗透压)causedbytheseadditionsleadstotheaccumulationofosmoprotectantsinthecell,whichstabilizethenativeproteinstructure.additionofethanol,lowmolecularweightthiolsanddisulfides,andNaCl.,3.Co-expressionofchaperonesand/orfoldases.Twoclassesofproteinsplayanimportantroleininvivoproteinfolding.Molecularchaperonespromotetheproperisomerizationandcellulartargetingbytransientlyinteractingwithfoldingintermediates.ThebestcharacterizedE.colisystemsare:GroES-GroELDnaK-DnaJ-GrpEClpB,GroEL/GroES复合物,GroES,GroEL,Foldasesacceleraterate-limitingstepsalongthefoldingpathway.Threetypesoffoldasesplayanimportantrole:peptidylprolylcis/transisomerases(PPIs肽基脯氨酰异构酶)disulfideoxidoreductase(DsbA)anddisulfideisomerase(DsbC)proteindisulfideisomerase(PDI)-aneukaryoticproteinthatcatalyzesbothproteincysteineoxidationanddisulfidebondisomerization.Italsoexhibitschaperoneactivity.,Co-expressionofoneormoreoftheseproteinswiththetargetproteincouldleadtohigherlevelsofsolubleprotein.Thelevelsofco-expressionofthedifferentchaperones/foldaseshavetobeoptimizedforeachindividualcase.DsbAandDsbChavealsoshownpossitiveeffectsonexpressionlevelswhenusedasafusionpartner.,4.Periplasmicexpression.Secretionofthetargetproteintotheperiplasmhasanumberofdistinctadvantages:theoxidizingenvironmentoftheperiplasmallowsfortheformationofdisulfidebonds,whichdoesnotoccurinthereducingenvironmentofthecytoplasm.theperiplasmcontainstwofoldases,disulfideoxidoreductase(DsbA)anddisulfideisomerase(DsbC),thatcatalyzetheformationandisomerizationofdisulfidebonds.reducedproteolysis(sincelessproteinsarepresent).allowsfortheaccumulationofproteinsthataretoxicinthecytoplasm.engineeringofanauthentic(可信的)N-terminus.,Secretionisachievedbytheadditionofaleadersequence(signalpeptide)totheN-terminusofthetargetprotein.MostusedleadersequencesarepelBandompT.Unfortunately,expressionyieldareusuallymuchlowerandnotallexpressedproteinissecretedintotheperiplasmbutisalsofoundinthemedium,thecytoplasmandthecytoplasmicmembrane.,5.Usingspecifichoststrains.Thesolubilityofdisulfidebondcontainingproteincanbeincreasedbyusingahoststrainwithamoreoxidizingcytoplasmicenvironment.Twostrainsarecommerciallyavailable(Novagen):AD494,whichhasamutationinthioredoxinreductase(trxB).Origami,adoublemutantinthioredoxinreductase(trxB)andglutathionereductase(gor).,6.Additionofafusionpartner.FusionoftheN-terminusofaheterologousproteintotheC-terminusofasolublefusionpartneroftenimprovesthesolubilityofthefusionprotein.7.Expressionofafragmentoftheprotein.E.colidoesnotexpresswellverylargeproteins(70kDa).Choosingasmallerfragmentofthetargetproteincanimproveexpressionlevelsandsolubility.Thesolubilityofapoorlysoluble(orinsoluble)proteincanalsobeimprovedbyselectingonlyasolubledomainforexpression.,8.Invitrodenaturationandrefoldingoftheprotein.Whendespitealleffortsthetargetproteinstillisexpressedininclusionbodies,thenthelastresortistodenatureandrefoldtheproteininvitro.Thisprocedureiscarriedoutinthreephases:isolationoftheinclusionbodies.solubilizationanddenaturationofthetargetprotein.Thisisdonebytheadditionofadenaturingagent(usuallyguanidineorurea)underreducingconditions(e.g.20mMDTT).refoldingoftheproteinbyremovingthedenaturatingagentusingdialysis,dilutionorchromatography.Forproteinscontainingdisulfidebondsthishastobecarriedoutinthepresenceofaredoxshuttlingsysteme.g.reducedandoxidizedglutathione.,3)如何改善蛋白质的稳定性,Ingeneralproperlyfoldedproteinarequitestable.Theprecisestructuralfeaturesthatimpartstabilitytoproteinsarenotknown.Nonetheless,somedeterminantsofproteininstabilityhavebeenelucidated.,1.N-endrule.InE.coliN-terminalArg,Lys,Leu,Phe,Tyr,andTrpresiduesgreatlydecreasethehalf-lifeoftheprotein.AtestproteinwiththeseresiduesintheN-terminalpositionshowedhalf-lifesofonly2minutescomparedtomorethan10hourswithallotheraminoacids(exceptproline).Reference:Tobiasetal.(1991)Science254,1374-1377.,2.PESThypothesis.IneukaryotesproteinsaredestabilizedbyregionsenrichedinPro,Glu,Ser,andThr.,Sometimesthelevelsofproteinexpressionarelowbecauseofhighlevelofproteaolyticdegradationofthetargetprotein.Thefollowingapproachescanbeusedtoimporvetheexpressionlevel:i.Usingprotease-deficienthoststrains.Theuseofhoststrainscarryingmutationswhicheliminatetheproductionofproteasescansometimesenhanceaccumulationbyreducingproteolyticdegradation.BL21,theworkhorseofE.coliexpression,isdeficientintwoproteasesencodedbythelon(cytoplasmic)andompT(periplasmic)genes.,ii.Periplasmicexpression.Intheperiplasmproteolyticdegradartionofproteinsisdecreased.Thisismainlybecausethetotalnumberofproteinsintheperiplasmislower.iii.Decreasingthegrowthtemperature.Reducingthegrowthtemperaturewillresultinslowerproteinproductionbutalsoinslowerproteolyticdegradation.Theoverallresultisoftenanincreasedexpressionlevelofthetargetprotein.,4）如何减少蛋白质的毒性,Lowexpressionlevelsornoexpressionatallcanalsobecausedbytoxicityofthetargetprotein.Proteintoxicitycanmanifestitselfondifferentlevels:,1.Incompleterepressionofproteinexpression.Manypromotersarenotverytightlyregulatedandshowsomedegreeofexpressionbeforetheadditionoftheinducer.Especiallylacpromotersareleaky.Oftenthisleakinessleadstoplasmidinstabilityand/orlossofplasmid.Asaconsequencetheculturewillbeovergrowbycellsthathavelosttheplasmid(especiallywhenampicillinisusedasaselectablemarker)orwillnotgrowatall.Differentapproachescanbeusedtogiveamoretightlyregulatedexpression:,constitutiveexpressionofarepressorprotein.InthecaseofthelacpromoterbytheexpressionofthelacrepressorfromthelacIorlacIqgene.FormediumcopynumberplasmidsitissufficienttouseahoststraincarryingthelacIqallele(等位基因).Highercopynumberplasmidsshouldcontaintherepressorgeneonthevector.useamoretightlyregulatedpromoter,e.g.thearabinosepromoter(PBAD).usealowercopynumberplasmid.,constitutiveexpressionofphageT7lysosymefromacompatiblepLysSorpLysEplasmid(Novagen).LysosymebindstoT7RNApolymeraseandinactivatestheenzyme.AftertheadditionofIPTGtheexpressionlevelofthepolymerasewillbemuchhigherthanthatoflysosymeandthiswillovercometherepression.additionof1%glucosetotheculturemediumtorepressinductionofthelacpromoterbylactose,whichispresentinmostrichmedia(suchasLB,2xYT).,2.Besidestighteningregulation,otherapproachesexisttopreventlosingtheexpressionvectorfromthecells.