细菌的基因表达课件

Gene expression of BacteriaContentsIntroductionGeneexpressionandregulationofbacteriaGenecloningandproteinexpressionIntroductionTypesoflifeProcaryotesEukaryotesVirusesThreekindomoforganismsTwotypesofbacteriaGrampositiveGramnegativeBacterialshapesBacterialcellstructureCapsulesFlagella and MotilityMotilityFimbriae and Sex piliGram-Positive Cell WallsGram-Negative Cell WallsThe Cytoplasmic MatrixGeneexpressionandregulation1.DNA,genesandchromosomesDNA(or deoxyribonucleic acid)isthemoleculethatcarriesthegeneticinformationinallcellularformsoflifeandsomeviruses.Itbelongstoaclassofmoleculescalledthenucleicacids,whicharepolynucleotides-thatis,longchainsofnucleotides.1)DNAanitrogenousbase:cytosine(C),guanine(G),adenine(A)orthymine(T)afive-carbonsugarmolecule(deoxyriboseinthecaseofDNA)aphosphatemoleculeEachnucleotideconsistsofthreecomponents:DoublehelixofDNAmoleculeGeneticcodonsAcodontablesetsouthowthetripletcodonscodeforspecificaminoacids.universaldegenerateTheenzymehelicasebreaksthehydrogenbondsholdingthetwostrandstogether,andbothstrandscanthenactastemplatesfortheproductionoftheoppositestrand.TheprocessiscatalysedbytheenzymeDNApolymerase,andincludesaproofreadingmechanism.DNAreplicationSemiconservative DNA ReplicationRolling-Circle Pattern of Replication.Bidirectional Replication.Rollingcirclereplicationproducesmultiplecopiesofasinglecirculartemplate2)GenesThe geneisthebasicphysicalandfunctionalunitofheredity.Itconsistsofaspecificsequenceofnucleotidesatagivenpositiononagivenchromosomethatcodesforaspecificprotein(or,insomecases,anRNAmolecule).ThestructuralcomponentsofageneCoding regions,called exons,which specify a sequence of amino acidsNon-coding regions,called introns,which do not specify amino acidsRegulatory sequences,which play a role in determining when and where the protein is made(and how much is made)GenesconsistofthreetypesofnucleotidesequenceA Bacterial Structural Gene.PromoterisaregionofDNAthatinitiatestranscriptionofaparticulargene.Promotersarelocatednearthetranscriptionstartsitesofgenes,onthesamestrandandupstreamontheDNA.Promoterscanbeabout1001000basepairslong.PromoterInbacteria,ThepromoterisrecognizedbyRNApolymeraseandanassociatedsigmafactor.Ineukaryotes:Theprocessismorecomplicated,andatleastsevendifferentfactorsarenecessaryforthebindingofanRNApolymeraseIItothepromoter.Promotersrepresentcriticalelementsthatcanworkinconcertwithotherregulatoryregions(enhancers,silencers,boundaryelements/insulators)todirecttheleveloftranscriptionofagivengene.1:RNAPolymerase,2:Repressor,3:Promoter,4:Operator,5:Lactose,6:lacZ,7:lacY,8:lacA.Corepromoter-theminimalportionofthepromoterrequiredtoproperlyinitiatetranscriptionIncludesthetranscriptionstartsite(TSS)andelementsdirectlyupstreamAbindingsiteforRNApolymeraseRNApolymeraseI:transcribesgenesencodingribosomalRNARNApolymeraseII:transcribesgenesencodingmessengerRNAandcertainsmallnuclearRNAsandmicroRNARNApolymeraseIII:transcribesgenesencodingtransferRNAsandothersmallRNAsGeneraltranscriptionfactorbindingsites,e.g.TATAboxProximalpromoter-theproximalsequenceupstreamofthegenethattendstocontainprimaryregulatoryelementsApproximately250basepairsupstreamofthestartsiteSpecifictranscriptionfactorbindingsitesDistalpromoter-thedistalsequenceupstreamofthegenethatmaycontainadditionalregulatoryelements,oftenwithaweakerinfluencethantheproximalpromoterPromoterelementsBacterial promotersInbacteria,thepromotercontainstwoshortsequenceelementsapproximately-10and-35nucleotidesupstreamfromthetranscriptionstartsite.Thesequenceat-10(the-10element)hastheconsensussequenceTATAAT.Thesequenceat-35(the-35element)hastheconsensussequenceTTGACA.Ingenetics,anenhancerisashort(50-1500bp)regionofDNAthatcanbeboundwithproteins(activators)toactivatetranscriptionofageneorgenes.Theseproteinsareusuallyreferredtoastranscriptionfactors.Enhancersaregenerallycis-acting,locatedupto1Mbp(1,000,000bp)awayfromthegeneandcanbeupstreamordownstreamfromthestartsite,andeitherintheforwardorbackwarddirectionTherearehundredsofthousandsofenhancersinthehumangenome.EnhancerSilencerIngenetics,asilencerisaDNAsequencecapableofbindingtranscriptionregulationfactors,calledrepressors.WhenarepressorproteinbindstothesilencerregionofDNA,RNAispreventedfrombindingtothepromoterregion.WiththetranscriptionofDNAintoRNAblocked,thetranslationofRNAintoproteinsisimpossible.Thus,silencerspreventgenesfrombeingexpressedasproteins.TerminatorIngenetics,atranscriptionterminatorisasectionofnucleicacidsequencethatmarkstheendofageneoroperoningenomicDNAduringtranscription.ThissequencemediatestranscriptionalterminationbyprovidingsignalsinthenewlysynthesizedmRNAthattriggerprocesseswhichreleasethemRNAfromthetranscriptionalcomplex.TheseprocessesincludethedirectinteractionofthemRNAsecondarystructurewiththecomplexand/ortheindirectactivitiesofrecruitedterminationfactors.ReleaseofthetranscriptionalcomplexfreesRNApolymeraseandrelatedtranscriptionalmachinerytobegintranscriptionofnewmRNAs.Rho-dependentterminatorsRho-independentterminatorsRhofactor,whichexhibitsRNAhelicaseactivity,disruptthemRNA-DNA-RNApolymerasetranscriptionalcomplexRho-independentterminatorsrequiretheformationofaself-annealinghairpinstructureontheelongatingtranscript,whichresultsinthedisruptionofthemRNA-DNA-RNApolymeraseternarycomplex.3)ChromosomesAchromosomeispackagedandorganizedchromatin,acomplexofmacromoleculesfoundincells,consistingofDNA,proteinandRNA.Chromosome unraveling to show the base pairings of the DNAChromosomal Organization in Bacteria and Viruses.2.GeneexpressionThecentraldogmaofmolecularbiology1)TranscriptionTranscription:Synthesizing RNA from a DNA TemplateRNA molecules are synthesized that are complementary and antiparallel to one of the two nucleotide strands of DNA,the template strand.RNA is transcribed from one DNA strand.AtranscriptionunitisapieceofDNAthatencodesanRNAmoleculeandthesequencesnecessaryforitspropertranscription.Eachtranscriptionunitincludesapromoter,anRNA-codingregion,andaterminator.Ribonucleoside triphosphates are substrates used in RNA synthesis.In transcription,nucleotides are alwaysadded to the 3 end of the RNA molecule.BacterialcellspossessasingletypeofRNApolymerase,consistingofacoreenzymeandothersubunitsthatparticipateinvariousstagesoftranscription.Type Transcribes RNApolymeraseILargerRNAsRNApolymeraseIIPre-mRNA,somesnRNAs,snoRNAsRNApolymeraseIIItRNAs,smallrRNA,snRNAsEukaryoticcellspossessthreedistincttypesofRNApolymeraseThe Process of Bacterial TranscriptionInitiation,inwhichthetranscriptionapparatusassemblesonthepromoterandbeginsthesynthesisofRNA;elongation,inwhichRNApolymerasemovesalongtheDNA,unwindingitandaddingnewnucleotides,oneatatime,tothe3endofthegrowingRNAstrand;termination,therecognitionoftheendofthetranscriptionunitandtheseparationoftheRNAmoleculefromtheDNAtemplate.In bacterial promoters,consensus sequences are found upstream of the start site,approximately at positions 10 and 35.InitiationInitiationInitiationterminationmRNA Transcription From DNA.Eucaryotic mRNA Synthesis.3)TranslationProteinsynthesiscanbedividedintofourstages:ThebindingofaminoacidstothetRNAs;Initiation,inwhichthecomponentsnecessaryfortranslationareassembledattheribosome;Elongation,inwhichaminoacidsarejoined,oneatatime,tothegrowingpolypeptidechain;Termination,inwhichproteinsynthesishaltsattheterminationcodonandthetranslationcomponentsarereleasedfromtheribosome.An amino acid attaches to the 3end of a tRNA.Certain positions on tRNA molecules are recognized by the appropriate aminoacyl-tRNA synthetase.The binding of amino acids to the tRNAsAn amino acid becomes attached to the appropriate tRNA in a two-step reactionInitiationThe initiation of translation in bacterialcells requires several initiation factors and GTP.Shine-Dalgarno consensus sequences in mRNA are required for the attachment of the small subunit of the ribosome.ElongationAribosomehasthreesitesthatcanbeoccupiedbytRNAs;theaminoacyl,orA,site,thepeptidyl,orP,site,andtheexit,orE,siteThe first step is the delivery of a charged tRNA(tRNA with its amino acid attached)to the A site.The second step of elongation is the creation of a peptide bond between the amino acids that are attached to tRNAs in the P and A sitesThe third step in elongation is translocation,the movement of the ribosome down the mRNA in the 5-3 direction.Elongationoccursinthreesteps.Translation ends when a stop codon isencountered.TerminationThe four steps involved in translation are tRNA charging(the binding of amino acids to tRNAs),initiation,elongation,and terminationIn prokaryotic cells,transcription and translation take place simultaneouslyThePosttranslationalModificationsofProteins3.Regulation of gene expressionThe giant transgenic mouse on the left was produced by injecting a ratgene for growth hormone into a mouse embryo;a normal-size mouse ison the right.General Principles of Gene RegulationOneofthemajorthemesofmoleculargeneticsisthecentraldogma,whichstatedthatgeneticinformationflowsfromDNAtoRNAtoproteinsandprovidedamolecularbasisfortheconnectionbetweengenotypeandphenotype.Althoughthecentraldogmabroughtcoherencetoearlyresearchinmoleculargenetics,itfailedtoaddressacriticalissue:Howistheflowofinformationalongthemolecularpathwayregulated?Inbacteria,generegulationmaintainsinternalflexibility,turninggenesonandoffinresponsetoenvironmentalchanges.Inmulticellulareukaryoticorganisms,generegulationbringsaboutcellulardifferentiation.Gene expression may be controlled atmultiple levels.GeneexpressionmaybecontrolledatanyofanumberofpointsalongthemolecularpathwayfromDNAtoprotein,includinggenestructure,transcription,mRNAprocessing,RNAstability,translation,andposttranslationalmodification.GenesandRegulatoryElementsGene Regulation in Bacterial CellsOperonFunctionallyrelatedgenesinbacterialcellsarefrequentlyclusteredtogetherasasingletranscriptionalunittermedanoperon.Atypicaloperonincludesseveralstructuralgenes,apromoterforthestructuralgenes,andanoperatorsitewheretheproductofaregulatorgenebinds.An operon is a single transcriptional unit that includes a series of structural genes,a promoter,and an operator.cataboliterepressionisatypeofpositivecontrolinthelac operon.Binding of the cAMPCAP complex to DNA produces a sharp bend in DNA that activatestranscription.trp operon controlsAttenuation:The Premature Termination of Transcription细菌的应急反应细菌的应急反应氨基酸匮乏空载tRNA焦磷酸转移酶ppGpp结合RNA聚合酶改变构型与启动子结合受阻结合启动子改变与RNA聚合酶结合改变转录通过通过因子更换的调控因子更换的调控枯草芽孢杆菌芽孢形成过程中枯草芽孢杆菌芽孢形成过程中因子的更换因子的更换 55，28，32，37，29大肠杆菌的热激应答大肠杆菌的热激应答Gene clone and expression1.Gene CloningThis describes the process of copying fragments of DNA which can then be used for many different purposes,such as creating GM crops,or finding a cure for disease.There are two types of gene cloning:in vivo,which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells.The other type is in vitro which is using the polymerase chain reaction(PCR)method to create copies of fragments of DNA.1)Gene cloning:in vivoDNA fragments isolation(flankingsequence)Gene Libraries construction(genomiclibrary,cDNAlibrary)Screen librariesClone and subcloneDNA fragments isolationMappingReversePCRTailPCRGene Libraries constructionAgenelibraryisalargecollectionofclonedDNAsequencesfromasinglegenome.Agenomiclibrary,intheory,wouldcontainatleastonecopyofeverysequenceinanorganismsgenome.Theseareusedtoinvestigatethestructureofagivenchromosome,ortoclonespecificgenes.Thesetypesoflibrariesmaybepreparedfromasubsetoftheentiregenome(forexample,asinglechromosome).Thefirststepincreatingagenomiclibraryistobreakup,orfractionate,thegenomeusingphysicalmethodsorrestrictionenzymes.Thefragmentsarethenlinkedtoappropriatevectorsandclonedinasuitablehostcellpopulation.AcDNAlibrary(complementaryDNA)containsDNApresentinagivencellpopulationwhichispreparedfromthemRNA(messengerRNA)usingtheenzymereversetranscriptase.TheresultingcDNArepresentsthegenesexpressedinthecellpopulationasasubsetoftheentiregenome,andcanbeclonedusingavectorandsuitablehostcell.ThecDNAwillnotincludeintronsorregulatorysequencesastheseareremovedfromtheRNAduringprocessing,andthismakesacDNAlibraryeasiertomaintain.AcDNAlibrarycanalsobepreparedusingreversetranscriptasePCR(RT-PCR).Screen librariesCloneandsubclone2)Gene cloning:in vitroPolymerasechainreactionPolymerasechainreactionDNA template that contains the DNA region(target)to be amplified.Two primers that are complementary to the 3(three prime)ends of each of the sense and anti-sense strand of the DNA target.Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C.Deoxynucleoside triphosphates(dNTPs),the building-blocks from which the DNA polymerase synthesizes a new DNA strand.Buffer solution,providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.Bivalent cations,magnesium or manganese ions;generally Mg2+is used,but Mn2+can be utilized for PCR-mediated DNA mutagenesis,as higher Mn2+concentration increases the error rate during DNA synthesisMonovalent cation potassium ions.PCRPrimerDesignGuidelinesPrimerLength(18-22bp)MeltingTemperature(TM),Primerswithmeltingtemperaturesintherangeof52-58generallyproducethebestresults.AnnealingTemperatureGCContent:TheGCcontentofprimershouldbe40-60%.GCClamp:ThepresenceofGorCbaseswithinthelastfivebasesfromthe3endofprimers(GCclamp)helpspromotespecificbindingatthe3endPrimerSecondaryStructuresRepeats3EndStabilityAvoidTemplateSecondaryStructureAvoidCrossHomologyDNA polymeraseTaqPfuFusion3.Gene expressionGeneexpressionistheprocessbywhichinformationfromageneisusedinthesynthesisofafunctionalgeneproduct.Theseproductsareoftenproteins,butinnon-proteincodinggenessuchas,transferRNA(tRNA)orsmallnuclearRNA(snRNA)genes,theproductisafunctionalRNA.1）ExpressionvectorAnexpressionvector,otherwiseknownasanexpressionconstruct,isusuallyaplasmidorvirusdesignedforproteinexpressionincells.Thevectorisusedtointroduceaspecificgeneintoatargetcell,andcancommandeerthecellsmechanismforproteinsynthesistoproducetheproteinencodedbythegene.Theplasmidisengineeredtocontainregulatorysequencesthatactasenhancerandpromoterregionsandleadtoefficienttranscriptionofthegenecarriedontheexpressionvector.Thegoalofawell-designedexpressionvectoristheproductionofsignificantamountofstablemessengerRNA,andthereforeproteins.ExpressionvectorsarebasictoolsforbiotechnologyandtheproductionofproteinsElements of expression vectorsoriginofreplicationselectablemarkermultiplecloningsiteA（inducible）strongpromoterTranslationinitiationsequencesuchasaribosomalbindingsiteorKozakconsensussequence.StartcodonStrongterminationcodonAtranscriptionterminationsequenceProteintags(histidinetag,glutathioneS-transferaseormaltose-bindingprotein)pAUR101是酿酒酵母是酿酒酵母（Saccharomyces cerevisiae）染色体整合型穿染色体整合型穿梭载体梭载体2）ProkaryoticexpressionsystemsEscherichia coliBacillus subtilisStreptomyces.sp原核表达系统的优点原核表达系统的优点细菌繁殖快，培养简单，价格低廉外源基因表达产物的水平高（最高达80%）表达特性清楚大肠杆菌表达系统的不足大肠杆菌表达系统的不足高效表达，易形成包涵体不能进行翻译后的加工修饰，如磷酸化、糖基化等大肠杆菌表达系统的操作流程大肠杆菌表达系统的操作流程获得目的基因获得目的基因准备表达载体准备表达载体将目的基因插入表将目的基因插入表达载体（测序验证）达载体（测序验证）转化表达宿主菌转化表达宿主菌诱导靶蛋白表达诱导靶蛋白表达表达蛋白的分析表达蛋白的分析表达蛋白的纯化表达蛋白的纯化大肠杆菌表达系统的几种表达方式大肠杆菌表达系统的几种表达方式组成型表达，表达载体的启动子为组成型启动子。组成型表达，表达载体的启动子为组成型启动子。成本低，表达量高，但不适合表达毒蛋白成本低，表达量高，但不适合表达毒蛋白诱导型表达，表达载体的启动子为组成型启动子，诱导型表达，表达载体的启动子为组成型启动子，诱导剂存在才能表达。另外一种类型是启动子是诱导剂存在才能表达。另外一种类型是启动子是组成型的，但是转录依赖的组成型的，但是转录依赖的RNARNA聚合酶是诱导型的。聚合酶是诱导型的。诱导型表达是蛋白表达的主流。诱导型表达是蛋白表达的主流。大肠杆菌表达系统的几种表达方式大肠杆菌表达系统的几种表达方式融合表达，表达载体多克隆位点上存在一段融合融合表达，表达载体多克隆位点上存在一段融合表达标签（表达标签（Tag)Tag)，表达产物为融合蛋白。方便后，表达产物为融合蛋白。方便后继的纯化或者检测。有时候能够提高蛋白的溶解继的纯化或者检测。有时候能够提高蛋白的溶解性。性。分泌表达，起始密码和目的基因加入信号肽，引分泌表达，起始密码和目的基因加入信号肽，引导蛋白穿过细胞膜进入周质空间，避免形成包涵导蛋白穿过细胞膜进入周质空间，避免形成包涵体。体。不可溶表达，强启动子导致表达出的蛋白来不及不可溶表达，强启动子导致表达出的蛋白来不及折叠，形成不可溶的包涵体。包涵体容易纯化，折叠，形成不可溶的包涵体。包涵体容易纯化，但是复性效率低。但是复性效率低。提高大肠杆菌表达系统重组蛋白可溶性的策略提高大肠杆菌表达系统重组蛋白可溶性的策略1 1）细胞中表达分子伴侣，辅助新生肽链折叠）细胞中表达分子伴侣，辅助新生肽链折叠（如（如TAKARATAKARA的的Chaperone plasmidChaperone plasmid）2 2）共表达折叠酶，催化共价键形成）共表达折叠酶，催化共价键形成3 3）降低培养温度和转速来降低蛋白合成速度）降低培养温度和转速来降低蛋白合成速度4 4）选择中等强度或低强度的启动子代替强启动子）选择中等强度或低强度的启动子代替强启动子5 5）选择适合的宿主菌株）选择适合的宿主菌株6 6）表达含可溶性多肽或信号肽的重组蛋白，提高可溶性）表达含可溶性多肽或信号肽的重组蛋白，提高可溶性。7 7）利用诱变技术改变氨基酸序列，提高蛋白的可溶性）利用诱变技术改变氨基酸序列，提高蛋白的可溶性8 8）诱导过程中加入化学物质，增加渗透压或改变培养基）诱导过程中加入化学物质，增加渗透压或改变培养基的的pHpHMerck（Novagen）pET表达载体系列pETpET系列载体是目前应用最为广泛的原核表达系统，系列载体是目前应用最为广泛的原核表达系统，已经成功在大肠杆菌中表达了各种各样的异源蛋已经成功在大肠杆菌中表达了各种各样的异源蛋白。白。pETpET系列载体是利用大肠杆菌噬菌体系列载体是利用大肠杆菌噬菌体T T7 7转录系转录系统进行表达的载体。统进行表达的载体。两类pET表达载体转录载体：用以表达本身带有原核核糖体结合位转录载体：用以表达本身带有原核核糖体结合位点和点和AUG AUG 起始密码子的目的基因。只有起始密码子的目的基因。只有3 3 种转录种转录载体：载体：pETpET-21(+)-21(+)、pETpET-24(+)-24(+)和和pET-23(+)pET-23(+)。翻译载体：包括来自翻译载体：包括来自 T7 T7 噬菌体主要衣壳蛋白的噬菌体主要衣壳蛋白的高效核糖体结合位点，用于表达那些不带有核糖高效核糖体结合位点，用于表达那些不带有核糖体结合位点的目的基因体结合位点的目的基因转录载体转录载体翻译载体翻译载体选择pET载体需要考虑的基本因素目的蛋白的应用目的蛋白已知特定信息克隆策略蛋白溶解性及细胞定位载体可以通过三种方式改善目的蛋白的溶解性或正确折叠：1)与本身溶解性高的多肽序列融合表达例如谷胱甘肽-S-转移酶(GST)，硫氧还蛋白(Trx)及NusA(NutilizationsubstanceA)。2)与催化二硫键形成的酶融合表达(例如Trx，DsbA，及DsbC)3)与信号序列融合表达，输出到细胞周质。适宜蛋白表达的大肠杆菌菌株的类型适宜蛋白表达的大肠杆菌菌株的类型1.蛋白酶缺陷型：所有的B菌株的衍生菌株都是lon蛋白酶和ompT蛋白酶缺陷型。包括BL21，B834，BLR,OrigamiTMB和TunerTM等。BL21(DE3)是应用最多的表达菌株。另外其衍生菌株BLR(DE3)蛇recA-，提高质粒在细菌中的稳定性。2.保证所有细胞以同样量表达：TunerTM菌株机器衍生菌株（OrigamiTMB和RosettaTM）是BL21菌株的lacY1缺陷突变株，这些菌株中所有细胞中蛋白以同样水平表达，对于诱导物如IPTG浓度的改变更加敏感。3.二硫键形成与溶解性增强：二硫键的形成有助于增加某些蛋白的可溶性，一些谷胱甘肽还原酶（gor）和/或硫氧还蛋白还原酶（trxB）缺陷型菌株如AD494，BL21trxB，Origami，OrigamiB，Rosetta-gamiTM等表达蛋白时候，可以更大程度促进二硫键的形成，提高蛋白的可溶性。4.稀有密码子的补给：Rosetta系列原核表达需要考虑的问题原核表达需要考虑的问题芽孢杆菌表达系统芽孢杆菌表达系统本系统具有如下优点：尤其适用于生产分泌型蛋白质。几乎不存在密码子的偏爱性生产有活性的蛋白质，尤其是真核起源的分泌型蛋白质一般具有S-S键结构，该系统也能进行具有活性的表达 易于培养和灭菌简单的基因技术安全的宿主菌。芽孢杆菌表达系统芽孢杆菌表达系统本系统具有如下不足质粒的稳定性差。转化效率低多是组成型表达，毒蛋白表达困难芽孢杆菌表达系统芽孢杆菌表达系统枯草芽孢杆菌（Bacillussubtilis）表达系统短小芽孢杆菌（Brevibacillus）表达系统。短小芽孢杆菌表达载体短小芽孢杆菌表达载体 枯草芽孢杆菌表达载体枯草芽孢杆菌表达载体3）真核表达系统酵母表达系统酵母表达系统酿酒酵母表达系统酿酒酵母表达系统甲醇酵母表达系统甲醇酵母表达系统其它酵母表达系统其它酵母表达系统酵母表达系统的产生 基因工程技术的发展为用微生物合成和生产外源蛋白展示出广阔的前景。长期以来，人们用大肠杆菌作为宿主表达了多种蛋白。大肠杆菌表达系统存在若干缺陷：A：缺少真核生物的蛋白翻译后修饰和加工B：表达的蛋白多以包含体形式存在，需要经过复杂的复性才能恢复构象和活性C：背景杂蛋白很多、纯化起来麻烦D：表达量一般不是很高1979年，为了克服大肠杆菌表达系统的缺点，发展了酵母表达系统。最先使用的是酿酒酵母。1981年,Hizeman等人首次报道了人重组干扰素基因在酿酒酵母中表达并获成功。酵母表达系统的优点A.可以大规模生产，有效降低了生产成本B.收获的外源蛋白质具有一定程度上的折叠加工和糖基化修饰，性质较原核表达的蛋白质更加稳定C.很容易纯化酵母表达系统的组成宿主质粒载体自主复制型质粒 选择标记外源基因表达的相关元件主要酵母表达系统酿酒酵母表达系统乳酸克鲁维亚酵母表达系统 甲醇营养型酵母表达系统裂殖酵母表达系统酿酒酵母表达系统酿酒酵母分泌系统酿酒酵母分泌系统酿酒酵母系统启动子酿酒酵母系统启动子酿酒酵母表达系统存在的问题酿酒酵母表达系统存在的问题酿酒酵母糖基化系统酿酒酵母糖基化系统1）糖酵解途径中关键酶的强启动子，受葡萄糖诱导：甘油醛甘油醛-3-磷酸脱氢酶基因磷酸脱氢酶基因GAPDH 磷酸甘油激酶基因磷酸甘油激酶基因PKG 乙醇脱氢酶基因乙醇脱氢酶基因ADH酿酒酵母表达系统常用启动子酿酒酵母表达系统常用启动子pho4pho4TSTS-PHO5-PHO5启动子启动子A、PHO5启动子在培养基缺磷酸盐时启动转录启动子在培养基缺磷酸盐时启动转录B、PHO4基因编码产物是基因编码产物是PHO5启动子的正调控因子启动子的正调控因子C C、pho4TS 温度敏感，温度敏感，35时失活，时失活，PHO5关闭关闭D、pho4TS-PHO5启动子通过降温（启动子通过降温（23）诱导表达）诱导表达低温诱导、磷酸盐抑制低温诱导、磷酸盐抑制酿酒酵母系统常用分泌信号肽来源：酿酒酵母系统常用分泌信号肽来源：性结合因子：性结合因子：MF-酸性磷酸酯酶：酸性磷酸酯酶：PHO5 蔗糖酶：蔗糖酶：SUC2 杀手毒素因子：杀手毒素因子：KIL酿酒酵母分泌系统*保守性低，大多异源宿主系统的信号肽不能保守性低，大多异源宿主系统的信号肽不能互用互用*信号肽结构：信号肽结构：酿酒酵母信号肽特点正电荷区正电荷区疏水区疏水区极性区极性区Met目的蛋白目的蛋白信号肽剪切位点信号肽剪切位点*分泌效率高分泌效率高*在酵母系统具有通用性在酵母系统具有通用性*88个残基组成个残基组成 MF-MF-信号肽信号肽Met目的蛋白目的蛋白-Lys-Arg-Glu-Ala-Glu-Ala-KEX2 DAPDAPDAP：STE13编码的二肽酶编码的二肽酶糖基化位点：Asn-X-Thr/Ser（X代表任何氨基酸）N-型糖基化：天门冬酰氨酸残基上的酰胺氮进行糖基化，对蛋白质的折叠、稳定性及活性较重要。O-型糖基化：苏氨酸/丝氨酸上的羟基氧进行糖基化 酿酒酵母糖基化系统酿酒酵母糖基化系统*过糖基化（超糖基化修饰）：N型或O型糖基的外链进一步形成庞大的、由甘露糖组成的、复杂分支结构的现象。增加了免疫原性、对活性与药代稳定性均有影响。*糖链组成O型糖链仅由甘露糖组成、而哺乳细胞的还含唾液酸基团酿酒酵母糖基化特点1）表达水平普遍不高A、表达载体传代不稳定（YEp、YRp）B、所采用的强启动子调控不严谨C、不能利用简单的无机培养基进行高密度发酵2）分泌表达产物过糖基化酿酒酵母表达系统的缺陷甲醇酵母表达系统甲醇酵母与甲醇氧化酶启动子甲醇酵母与甲醇氧化酶启动子甲醇酵母表达系统的应用甲醇酵母表达系统的应用甲醇酵母表达系统操作原理甲醇酵母表达系统操作原理甲醇酵母系统高效表达影响因素与对策甲醇酵母系统高效表达影响因素与对策甲醇酵母表达系统的优缺点甲醇酵母表达系统的优缺点甲醇酵母与甲醇氧化酶启动子甲醇酵母（甲醇酵母（methylotrophic yeast)指可利用甲醇作单一碳源的一类酵母。指可利用甲醇作单一碳源的一类酵母。毕赤酵母（毕赤酵母（Pichia pastoris)汉森酵母（汉森酵母（Hansenula ploymorpha)假丝酵母（假丝酵母（Candia boidinii)甲醇酵母与甲醇氧化酶启动子甲醇氧化酶甲醇氧化酶A、甲醇代谢关键酶，占可溶蛋白的、甲醇代谢关键酶，占可溶蛋白的30%以上以上 B、名称和拷贝数不同、名称和拷贝数不同毕赤酵母毕赤酵母/假丝酵母假丝酵母 汉森酵母汉森酵母 醇氧化酶 甲醇氧化酶alcohol oxidase,AOX methanol oxidase,MOXAOX1、AOX2 MOX 甲醇酵母与甲醇氧化酶启动子AOX1与AOX2*毕赤酵母和假丝酵母基因组存在二个毕赤酵母和假丝酵母基因组存在二个AOX基因基因 AOX1、AOX2*AOX1与与AOX2基因基因97%同源同源*AOX1 占主导地位，负责占主导地位，负责AOX 99%以上活性以上活性甲醇酵母与甲醇氧化酶启动子甲醇氧化酶启动子甲醇氧化酶启动子A、目前已发现的、最强的真核启动子、目前已发现的、最强的真核启动子B、严谨调控型启动子、严谨调控型启动子 AOX1：葡萄糖和甘油脱阻遏、甲醇诱导：葡萄糖和甘油脱阻遏、甲醇诱导 MOX：葡萄糖阻遏、甘油脱阻遏、甲醇诱导：葡萄糖阻遏、甘油脱阻遏、甲醇诱导 甲醇酵母表达系统的优缺点A、表达水平高（最高水平的系统）、表达水平高（最高水平的系统）B、产物可翻译后修饰：糖基化、磷酸化、酰脂化、产物可翻译后修饰：糖基化、磷酸化、酰脂化C、过糖基化程度比酿酒酵母少（、过糖基化程度比酿酒酵母少（8-15个个vs100-150 个甘露糖）个甘露糖）D、产物可正确折叠和高效分泌（最高分泌表达系统）、产物可正确折叠和高效分泌（最高分泌表达系统）E、可利用简单无机盐培养基高密度发酵，生物量大。、可利用简单无机盐培养基