大腸桿菌中蛋白質(zhì)分泌及雙元表達(dá)機(jī)制

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

to

ExtracellularItype（α-HlyA）HlyB、HlyD、TolCIItypeMechanismsofProteinSecretionTypeISecretion

to

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

Post-translationalpathwayTatpathwaySecpathwayComparationofSecpathwayandTatpathwayco-translationalpathwayAnoverviewofco-translationalproteintargetingbythebacterialSRPSRPandSRaremulti-stateregulatoryGTPasesthatundergoaseriesofconformationalchangesduringtheirGTPasecycleConformationalchangesinSRPandSRGTPasesarecoupledtoglobalreorganizationoftheSRPparticleandareregulatedbybiologicaleffectorsforthepathway

SecretoryExpressionofRecombinantProteinSignalpeptidesequenceCo-expressionFusionexpressionExpressionhostSignalPeptideSequence增強(qiáng)信號(hào)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(qiáng)信號(hào)肽的外分泌強(qiáng)度信號(hào)肽N端的PI值和親水性與采取通過何種分泌途徑有關(guān)信號(hào)肽N端編碼區(qū)的RNA自由能GRNA不僅影響翻譯效率，也會(huì)影響Tat途徑的轉(zhuǎn)運(yùn)效率。N區(qū)疏水H區(qū)增加信號(hào)肽疏水區(qū)的長(zhǎng)度和疏水性有利于重組蛋白表達(dá)。信號(hào)肽加工C區(qū)改變信號(hào)肽C區(qū)末尾切割位點(diǎn)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(nèi)啡肽融合OmpF后可以將其分泌到胞外物理化學(xué)機(jī)械法超聲，溶氧量，pH，添加甘氨酸，酶制劑等手段增加細(xì)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(zhì)空間，部分定位在胞外，影響了分泌表達(dá)水平，II型在重組蛋白的規(guī)模化應(yīng)用方面仍有相當(dāng)?shù)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(shí)需要共表達(dá)特異性轉(zhuǎn)運(yùn)蛋白HlyB