大腸桿菌中蛋白質分泌及雙元表達機制

MechanismsofProteinSecretionandSecretoryExpressionofRecombinantProteininE.coli谷晨光Content1.Introduction2.MechanismsofProteinSecretion3.SecretoryExpressionofRecombinantProtein4.SummaryIntroductionSecretion

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ExtracellularItype（α-HlyA）HlyB、HlyD、TolCIItypeMechanismsofProteinSecretionTypeISecretion

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ExtracellularItype（α-HlyA）HlyB、HlyD、TolCIItypeProteintargetinginbacteriainnermembranePost-translationalpathwaysCo-translationalpathwayAschematicdepictionofvarioustargetingpathwaysfordeliveringproteinstothebacterialinnermembrane

Post-translationalpathwayTatpathwaySecpathwayComparationofSecpathwayandTatpathwayco-translationalpathwayAnoverviewofco-translationalproteintargetingbythebacterialSRPSRPandSRaremulti-stateregulatoryGTPasesthatundergoaseriesofconformationalchangesduringtheirGTPasecycleConformationalchangesinSRPandSRGTPasesarecoupledtoglobalreorganizationoftheSRPparticleandareregulatedbybiologicaleffectorsforthepathway

SecretoryExpressionofRecombinantProteinSignalpeptidesequenceCo-expressionFusionexpressionExpressionhostSignalPeptideSequence增強信號肽N端的正電荷有利于重組蛋白的分泌率將起始密碼子改為比較稀有的纈氨酸增強信號肽的外分泌強度信號肽N端的PI值和親水性與采取通過何種分泌途徑有關信號肽N端編碼區(qū)的RNA自由能GRNA不僅影響翻譯效率，也會影響Tat途徑的轉運效率。N區(qū)疏水H區(qū)增加信號肽疏水區(qū)的長度和疏水性有利于重組蛋白表達。信號肽加工C區(qū)改變信號肽C區(qū)末尾切割位點的氨基酸可以提供重組蛋白的外分泌效率。RecentexamplesfusionproteinexpressionforextracellarproteinproductioninE.coilCo-expressionFusionexpression將人β-內啡肽融合OmpF后可以將其分泌到胞外物理化學機械法超聲，溶氧量，pH，添加甘氨酸，酶制劑等手段增加細胞外膜的通透性熱休克，凍融等方法Summary1.重組蛋白常常部分定位在周質空間，部分定位在胞外，影響了分泌表達水平，II型在重組蛋白的規(guī)?；瘧梅矫嫒杂邢喈數木窒扌?。I型可分泌蛋白范圍廣（50-4000氨基酸）2.在使用I型分泌途徑分泌外源蛋白時需要共表達特異性轉運蛋白HlyB