构建胰岛素基因工程菌

构建胰岛素工程菌前言：胰岛素的发现胰岛素的结构重组人胰岛素及类型重组人胰岛素基因工程菌的构建：Constructionoftheecotin(大肠杆菌素)–proinsulinexpressionplasmidConstructionofpEGP1plasmid胰岛素的发现1922FrederickBantingCharlesBest人胰岛素一级结构CHAINA:21aminoacidsCHAINB:30aminoacids胰岛素立体结构1翻译后不需要糖基化修饰，在细菌内合成的胰岛素具有完整的生物活性。2胰岛素是一个相对较小的分子，由两条多肽链组成，一条含21个氨基酸（A链）和另一条含30个氨基酸（B链）。胰岛素具有两个利于通过DNA重组技术生产的特点：重组人胰岛素1982年10月在美国上市，全球第一个商业化基因重组人胰岛素。1.Humulin（优泌林）2.Novolin（诺和灵）EliLillyandCompanyNovoNordisk欧洲市场的重组胰岛素类似物重组人胰岛素类型1.重组前胰岛素ChainBChainCChainA2.A,B链分开表达+ChainBChainA3.B-miniC-A重组表达ChainBChainAMiniChainC4.B-XXX-A重组表达X为氨基酸ChainBChainAX1X2XnN≤3重组人胰岛素基因工程菌的构建重组前胰岛素ChainBChainCChainA人胰岛素原表达法基因工程菌的构建战略：tacb-GalCpeptideoriAprMetMetMMNCBpeptideApeptide人胰岛素原的cDNA重组人胰岛素原转化分离纯化人胰岛素原表达法表达产物的后处理路线：SSSSCN胰蛋白酶CpeptideApeptideBpeptideMRRKRCNBrRSSSSCNNCSSSSR羧肽酶BB链中第22位上的Arg和第29位上的Lys由于良好折叠的原因，对胰蛋白酶不敏感人胰岛素原表达法获取人胰岛素cDNA，克隆在含有Ptac和b-gal基因的表达型载体上，两基因通过Met的密码子连接通过CNBr裂解法回收胰岛素原蛋白体外折叠胰蛋白酶去除C链，余下完整的A链和C末端带一Arg的B链羧肽酶获得人胰岛素结果：由于有C链的存在，胰岛素原在体外能形成天然的空间构象，使得正确折叠率达80％，成本为$50/gMaterials1.RestrictionenzymesfromNEBandMBIFermentas2.PCRpurificationandgelextractionkitsfromQiagen3.pCR-BluntII-TOPOkitandfromInvitrogenTop10competentcells4.E.ColiBL21(DE3)GoldfromStratagene5.E.coliGM2163(dam-anddcm-)fromNEB6.E.ColiSF120fromProf.GeorgeGeorgiou’slaboratory7.BovinetrypsinogenandthrombinfromSigma8.HisTrapFFcrudeandfromAmershamHiTrapNHS-activatedHPcolumns9.AntibodiesusedforELISAfromRoche10.StandardHumanproinsulinfromNIBSC11.MaxisorpRELISAplatesfromNunc,Copenhagen.12.ProteinmassstandardPeqGoldusedforSDS–PAGEfromPeqlab13.Mark12TMmarkerfromInvitrogen14.NuPAGE(4–12%)Bis–TrisgelsfromInvitrogen15.ZipTipC18fromMillipore16.MicroBCATMproteinassaykitfromPierce17.RP-HPLCNucleosilC18columnfromMachereyNagel,GermanyFrompage11to13and15AjamaluddinMalik,MarcoJenzsch,AndreasLubbert,RainerRudolph,BrigitteSohlingProteinExpressionandPurification55(2007)100–111Constructionoftheecotin–proinsulinexpressionplasmidpET20b-proinsulinThesourceofproinsulingeneforwardprimerreverseprimerPCRpCR-BluntII-TOPOkitsequencetransformE.ColiGM2163(dam-anddcm-)pEGP1BspEISalI(containingtheecotin-pepsinogenfusionproteingene)(胃蛋白酶原)thepepsinogenfragmentwasremovedencodedE.coliecotintransformE.coliGM2163(dam-anddcm-)pEG-PIdigestBspEISalIdigestdephosphorylateandpurify.purify.ligateE.coliBL21(DE3)GoldpET-20bFromforwardprimer5’-GGTTCCGGATCTGGTTCTGGTTCTCTGGTCCCCGlySerGlySerGlySerGlySerLeuValProCGCGGTAGTCACCACCACCACCACCACCGTTTTGTGArgGlySerHisHisHisHisHisHisArgAACCAACACCTGTGCGGC-3’Proinsulinreverseprimer5’-AGTGTCGACTTAGTTGCAGTAGTTCTCCAGCTGGTA-3’TerProinsulinTCCGGAGTCGACBspEISalIsequenceofprimerSchematicpresentationoftheecotin-proinsulinfusionproteinclonedinpTrc99a1-21：E.coliecotinsignalsequence22-162：E.coliecotin163-174：(GlySer)6linker175-180:LVPRGSthrombin(凝血酶)cleavagesite181-186:(His)6187:Argtrypsin(胰蛋白酶)cleavagesite188-273:ProinsulinpCR®-BluntIITOPO®RestrictionenzymesBspEI5'...T^CCGGA...3'3'...AGGCC^T...5'SalI5'...G^TCGAC...3'3'...CAGCT^G...5'EcoRI5'...G^AATTC...3'3'...CTTAA^G...5'E.coliGM2163(dam-anddcm-)damdcm:甲基转移酶保护DNA序列不被甲基化，提高限制性内切酶正确识别酶切位点的机率E.coliBL21(DE3)GoldMoreLikeaMammalproduceanincreasedamountofrareE.colitRNAsthatcorrespondtocodonsusedmorefrequentlybyotherorganisms.Thismodificationovercomesexpressionproblemsduetocodonbias[1].提高E.colitRNAs结合在其他生物体里广泛使用但在E.coli中很少使用的密码子的机率。克服了密码子偏爱性造成的表达问题。pEGP1pTrc99a[2]pTrc-eco-peps(abbreviatedaspEGP1)ExpresspepsinogenfusedtoE.coliecotin[1][1]:vorgelegtderAnovelstrategyfortheperiplasmicproductionofheterologousproteinsinE.coliT7表达系统IPTG诱导CurrentProtocolsinMolecularBiologyJohnWileyandSons,Inc.Chapter1Escherichiacoli,Plasmids,andBacteriophagesSectionIIVectorsDerivedfromPlasmidsUnit1.5IntroductiontoPlasmidBiologyFigure1.5.11ConstructionofpEGP1plasmidFrompage23topage26E.coliJM83ThesourceofecotingenePCRpHQPEX-30-5[1]5’end(Gly-Ser)3Primerforwardprimer3’endofpepsinogen(His)6primerreverseprimerPCRforwardprimerThesourceofpepsinogenpCR-BluntII-TOPOkitEcoRI-BspEIBspEI-SalIpurifiedbyagarosegelelectrophoresis(琼脂糖凝胶电泳)pTrc99aEcoRI-SalIdephosphorylatedligatepEGP1pTrc-eco-peps(A)Structuralmodeloftheecotindimer.(B)Schematicdrawoftheecotin-pepsinogenfusionproteinaspresentinpEGP1.1–20:ecotinsignalpeptide21-162:matureecotin163-174:GS6linker175-546:maturepepsinogen546-552:His65’-TTCTAAGAATTCGAAGGAGATATACATAATGAAGACCATTCTACCTGCA-3’ecotinsignalpeptidesequenceofprimer3’endofecotin-(Gly-Ser)3primerreverseprimerforwordprimer5’-ATCTTATCCGGAACCAGAACCAGAGlySerGlySerGlySerACCACGAACTACCGCATTGTCAATTT-3’Glyecotin5’end(Gly-Ser)3primerforwordprimer5’-GTATATGTCGACTTAGTGGTGGTGGTGTerHisHisHisHisGTGGTGAGCCACGGGGGCCAGACC-3’HisHispepsinogen5’-GTTATATCCGGATCTGGTTCTGGTTCTGGTSerGlySerGlySerGlySerGlyTACAAGGTCCCCCTCA-3’pepsinogen3’endofpepsinogen(His)6primerreverseprimerGAATTCTCCGGAGTCGACBspEIEcoRISalIE.coliecotingeneGENBANKACCESSION:M60876J057571cgtagaggatcaaaagagtagcgggaagcgtggcaaaaacgggcttttgctcacatttca61aattggttataaatatatttatatagcgattgattcaccagagatatttctgctggtttg121ctctctcattagaatttaacactaaaagagcaggtaaaattgtctgaatgttctttaagt181tattcataaagcaaattaataaatctgatgaatatgttaaccttc