重组胰岛素知识专家讲座

重组胰岛素RecombinantHumanInsulin秦斌2023.10.301第1页前言：胰岛素旳发现胰岛素旳构造重组人胰岛素及类型重组人胰岛素基因工程菌旳构建：Constructionoftheecotin(大肠杆菌素)–proinsulinexpressionplasmidConstructionofpEGP1plasmid2第2页胰岛素旳发现1922FrederickBantingCharlesBest[1].SimonHeller,PlamenKozlovski,PeterKurtzhalsDiabetesResearchandClinicalPractice78(2023)149–158[1][1]3第3页人胰岛素一级构造CHAINA:21aminoacidsCHAINB:30aminoacids4第4页胰岛素立体构造5第5页1.Humulin（优泌林）

重组人胰岛素2.Novolin（诺和灵）

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。EliLillyandCompanyNovoNordisk6第6页欧洲市场旳重组胰岛素类似物SimonHeller,PlamenKozlovski,PeterKurtzhalsDiabetesResearchandClinicalPractice78(2023)149–1587第7页重组人胰岛素类型1.重组前胰岛素ChainBChainCChainA2.A,B链分开体现+ChainBChainA冯佑民张友尚<<生物工程进展>>1998,Vol.16,No.48第8页3.B-miniC-A重组体现ChainBChainAMiniChainC4.B-XXX-A重组体现X为氨基酸ChainBChainAX1X2Xn

N≤3冯佑民张友尚<<生物工程进展>>1998,Vol.16,No.49第9页重组人胰岛素基因工程菌旳构建重组前胰岛素ChainBChainCChainA10第10页Materials1.RestrictionenzymesfromNEBandMBIFermentas2.PCRpurificationandgelextractionkitsfromQiagen3.pCR-BluntII-TOPOkitandfromInvitrogenTop10competentcells4.E.ColiBL21(DE3)GoldfromStratagene5.E.coliGM2163(dam-anddcm-)fromNEB

6.E.ColiSF120fromProf.GeorgeGeorgiou’slaboratory7.BovinetrypsinogenandthrombinfromSigma8.HisTrapFFcrudeandfromAmershamHiTrapNHS-activatedHPcolumns9.AntibodiesusedforELISAfromRoche10.StandardHumanproinsulinfromNIBSC11.MaxisorpRELISAplatesfromNunc,Copenhagen.12.ProteinmassstandardPeqGoldusedforSDS–PAGEfromPeqlab13.Mark12TMmarkerfromInvitrogen14.NuPAGE(4–12%)Bis–TrisgelsfromInvitrogen15.ZipTipC18fromMillipore16.MicroBCATMproteinassaykitfromPierce17.RP-HPLCNucleosilC18columnfromMachereyNagel,GermanyFrompage11to13and15

AjamaluddinMalik,MarcoJenzsch,AndreasLubbert,RainerRudolph,BrigitteSohlingProteinExpressionandPurification55(2023)100–11111第11页Constructionoftheecotin–proinsulinexpressionplasmidpET20b-proinsulinThesourceofproinsulingeneforwardprimerreverseprimerPCRpCR-BluntII-TOPOkitsequencetransformE.ColiGM2163(dam-anddcm-)12第12页pEGP1BspEISalI(containingtheecotin-pepsinogenfusionproteingene)

(胃蛋白酶原)thepepsinogenfragmentwasremovedencodedE.coliecotintransformE.coliGM2163(dam-anddcm-)pEG-PIdigestBspEISalIdigestdephosphorylateandpurify.purify.ligateE.coliBL21(DE3)Gold13第13页pET-20bFrom14第14页forwardprimer5’-GGT

TCC

GGA

TCT

GGT

TCT

GGT

TCT

CTGGTCCCC

GlySerGlySerGlySer

GlySer

LeuVal

Pro

CGCGGTAGTCACCACCACCACCACCACCGT

TTTGTG

ArgGlySerHis

HisHisHisHisHis

Arg

AACCAACACCTGTGC

GGC-3’

Proinsulinreverseprimer5’-AGTGTCGACTTAGTTGCAGTAGTTCTCCAGCTG

GTA-3’

Ter

ProinsulinTCC

GGAGTCGACBspEISalIsequenceofprimer15第15页AjamaluddinMalik,MarcoJenzsch,AndreasLübbert,RainerRudolph,BrigitteSöhlingManuscriptunderrevision-ProteinExpressionandPurification1-21：E.coliecotinsignalsequence22-162：E.coliecotin163-174：(GlySer)6

linker175-180:LVPRGSthrombin(凝血酶)cleavagesite181-186:(His)6187:Argtrypsin(胰蛋白酶)cleavagesite188-273:ProinsulinSchematicpresentationoftheecotin-proinsulinfusionproteinclonedinpTrc99a16第16页pCR®-BluntIITOPO®17第17页TOPOCloningTechnology18第18页RestrictionenzymesBspEI5'...T^CCGGA...3'

3'...AGGCC^T...5'

SalI5'...G^TCGAC...3'

3'...CAGCT^G...5'

EcoRI5'...G^AATTC...3'

3'...CTTAA^G...5'

19第19页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中很少使用旳密码子旳机率。克服了密码子偏爱性导致旳体现问题。E.coliGM2163(dam-anddcm-)

damdcm:甲基转移酶保护DNA序列不被甲基化，提高限制性内切酶对旳辨认酶切位点旳机率[1]from20第20页pEGP1pTrc99acloningvector[2]Sequence4176BP;1009A;1059C;1118G;990T;0other;pTrc99AfrompTrc98A&pUC18[2]pTrc-eco-peps(abbreviatedaspEGP1)ExpresspepsinogenfusedtoE.coliecotin[1][1]:vorgelegtderAnovelstrategyfortheperiplasmicproductionofheterologousproteinsinE.coliT7体现系统IPTG诱导21第21页CurrentProtocolsinMolecularBiologyJohnWileyandSons,Inc.Chapter1Escherichiacoli,Plasmids,andBacteriophagesSectionIIVectorsDerivedfromPlasmidsUnit1.5IntroductiontoPlasmidBiologyFigure1.5.1122第22页ConstructionofpEGP1plasmidFrompage23topage26E.coliJM83Thesourceofecotingene3’endofecotin-(Gly-Ser)3primerreverseprimerPCRpHQPEX-30-5[1]5’end(Gly-Ser)3Primerforwardprimer3’endofpepsinogen(His)6primerreverseprimerPCRforwardprimerThesourceofpepsinogenFrompage23topage26AjamaluddinMalik,RainerRudolph,BrigitteSöhlingProteinExpressionandPuriWcation47(2023)662–671[1]:ProvidedbyR.Bolli,ZLBBioplasmaAG,Bern,Switzerland23第23页pCR-BluntII-TOPOkitEcoRI-BspEIBspEI-SalIpurifiedbyagarosegelelectrophoresis(琼脂糖凝胶电泳)pTrc99aEcoRI-SalIdephosphorylatedligate

pEGP1pTrc-eco-peps24第24页(A)Structuralmodeloftheecotindimer.(B)Schematicdrawoftheecotin-pepsinogenfusionproteinaspresentinpEGP1.1–20:ecotinsignalpeptide21-162:matureecotin163-174:GS6linker175-546:maturepepsinogen546-552:His625第25页5’-TTCTAAGAATTCGAAGGAGATATACATAAT

GAAGACCATTCTACCTGCA-3’

ecotinsignalpeptidesequenceofprimer3’endofecotin-(Gly-Ser)3

primerreverseprimerforwordprimer5’-ATCTTATCCGGAACCAGAACCAGA

Gly

SerGly

Ser

GlySerACCACGAACTACCGCATTGTCAATTT-3’Glyecotin5’end(Gly-Ser)3primerforwordprimer5’-GTATATGTCGAC

TTAGTGGTGGTGGTG

Ter

HisHisHisHisGTGGTGAGCCACGGGGGCCAGACC-3’

HisHispepsinogen5’-GTTATATCCGGATCTGGTTCTGGTTCTGGT

Ser

GlySerGlySerGlySerGly

TACAAGGTCCCCCTCA-3’

pepsinogen3’endofpepsinogen(His)6primerreverseprimerGAATTCTCCGGAGTCGACBspEIEcoRISalI26第26页E.coliecotingeneGENBANKACCESSION:M60876J05757

1cgtagaggatcaaaagagtagcgggaagcgtggcaaaaacgggcttttgctcacatttca61aattggttataaatatatttatatagcgattgattcaccagagatatttctgctggtttg121ctctctcattagaatttaacactaaaagagcaggtaaaattgtctgaatgttctttaagt181tattcataaagcaaattaataaatctgatgaatatgttaaccttcagcgacatcatcggt241gaaaacctataaatgaagaaggaaagcaaa

aaaatgaaga

ccattctacctgcagtattg301tttgccgctttcgctaccac

ttccgcctgg

gcggcagaaagcgtccagccactggaaaaa

361atcgcgccttatccacaagctgaaaaagggatgaagcgtcaggtgattcagttaaccccg

421caagaagatgaatctaccctgaaagtagaactgttaatcggtcagacgctggaagtcgat481tgcaatttgcatcgtctcggcgggaagctggaaaacaaaacgctggaaggctggggctat

541gattattatgtctttgataaagtcagttccccggtttcaacgatgatggcctgcccggat

601ggcaagaaagagaagaaatttgtcaccgcgtatctgggcgatgctggaatgctgcgttac

661aacagcaagctgccgatcgtggtgtatacgccagacaatgtagatgtgaagtaccgcgtc

721tggaaggcggaagagaaaattgacaacgcg

gtagttcgctaaactgccgtgaagtgcggc781accccgtaggtcagacaaggcggtcagcgatccgacatccaacgcccgagccggttgcct841gatgcgacgctggccgtcttatcaggcctacaccgctgtgaagtgcggcaccccgtaggt901cagacaaggcggtcacgcgcatccgacatccaacgcccgagccggttgcctgatgcgacg961ctggcctcttatcaggcctacaccgctgtgaagtgctccaccccgtaggtcggataaggc1021ggtagcgatccgacatctaacgcaagccgttgcecotinsignalpeptidemature

ecotin27第27页E.coliecotin1.Ecotinhasnoroleinthehostmetabolism.Duetoitsrelativelysmallsize,themetabolicburdenislow[1].2.Prokaryoticsignalpeptidetransportthefusionproteins

totheperiplasm(周质)[2].3.Playanimportantroleintranslocation[2]

.4.Ecotinisabroad-rangeserine-proteaseinhibitor，whenrattrypsinogenwasrecombinantlyproducedandsecretedintotheperiplasmofE.coli,itformedarelativelytightandspecificcomplexwithE.coliecotinatneutralpH[1].5.Duetoastrongaffinityofecotinfortrypsinaswellastrypsinogen，trypsinogenorinactivetrypsinvariantsimmobilizedtoacolumncanserveasahighlyselectiveaffinitymaterial[2].引导分泌避免水解固定化亲和层析

[1]:AjamaluddinMalik,MarcoJenzsch,AndreasLubbert,RainerRudolph,BrigitteSohlingProteinExpressionandPurification55(2023)100–111[2]:vorgelegtderAnovelstrategyfortheperiplasmicproductionofheterologousproteinsinE.coli代谢负荷低28第2