1重组胰岛素重组胰岛素Recombinant Human Insulin秦斌秦斌2前言：前言：胰岛素的发现胰岛素的发现胰岛素的结构胰岛素的结构重组人胰岛素及类型重组人胰岛素及类型重组人胰岛素基因工程菌的构建：重组人胰岛素基因工程菌的构建：Construction of the ecotin(大肠杆菌素大肠杆菌素)proinsulin expression plasmidConstruction of pEGP1 plasmid3胰岛素的发现胰岛素的发现1922 Frederick Banting Charles Best1. Simon Heller , Plamen Kozlovski , Peter KurtzhalsDiabetes Research and Clinical Practice 78 (2007) 149158114人胰岛素一级结构人胰岛素一级结构CHAIN A: 21 amino acidsCHAIN B: 30 amino acids5胰岛素立体结构胰岛素立体结构61.Humulin（优泌林）（优泌林） 重组人胰岛素重组人胰岛素2.Novolin（诺和灵）（诺和灵） 1982年年10月在美国上市，全球第一月在美国上市，全球第一个商业化基因重组人胰岛素。个商业化基因重组人胰岛素。Eli Lilly and Company Novo Nordisk 7欧洲市场的重组胰岛素类似物欧洲市场的重组胰岛素类似物Simon Heller , Plamen Kozlovski , Peter KurtzhalsDiabetes Research and Clinical Practice 78 (2007) 1491588重组人胰岛素类型重组人胰岛素类型1.重组前胰岛素重组前胰岛素Chain BChain CChain A2.A,B链分开表达链分开表达 +Chain BChain A冯佑民 张友尚 1998, Vol.16, No. 493.B-miniC-A 重组表达重组表达Chain BChain AMini Chain C4.B-XXX-A 重组表达重组表达 X为氨基酸为氨基酸 Chain BChain AX1X2Xn N3冯佑民 张友尚 1998, Vol.16, No. 410重组人胰岛素基因工程菌的构建重组人胰岛素基因工程菌的构建重组前胰岛素重组前胰岛素Chain BChain CChain A11Materials1.Restriction enzymes from NEB and MBI Fermentas2.PCR purification and gel extraction kits from Qiagen3.pCR-Blunt II-TOPO kit and from Invitrogen Top10 competent cells4.E. Coli BL21(DE3)Gold from Stratagene5.E. coli GM2163 (dam- and dcm- ) from NEB 6.E. Coli SF120 from Prof. George Georgious laboratory7.Bovine trypsinogen and thrombin from Sigma8.HisTrap FF crude and from Amersham HiTrap NHS-activated HP columns 9. Antibodies used for ELISA from Roche10. Standard Human proinsulin from NIBSC 11. MaxisorpR ELISA plates from Nunc,Copenhagen.12. Protein mass standard PeqGold used for SDSPAGE from Peqlab13. Mark12 TM marker from Invitrogen14. NuPAGE (412%) BisTris gels from Invitrogen15.ZipTipC18 from Millipore16. Micro BCATM protein assay kit from Pierce17. RP-HPLC Nucleosil C18 column from Macherey Nagel, GermanyFrom page 11 to 13 and 15 Ajamaluddin Malik , Marco Jenzsch , Andreas Lubbert ,Rainer Rudolph , Brigitte Sohling Protein Expression and Purification 55 (2007) 10011112Construction of the ecotinproinsulin expression plasmid pET 20b-proinsulin The source of proinsulin geneforward primerreverse primerPCRpCR-Blunt II-TOPO kitsequencetransformE. Coli GM2163(dam- and dcm- )13pEGP1BspE ISal I(containing the ecotin-pepsinogen fusion protein gene) (胃蛋白酶原)the pepsinogen fragment was removed encoded E. coli ecotintransformE. coli GM2163 (dam- and dcm- )pEG-PIdigestBspE ISal Idigestdephosphorylate and purify.purify.ligateE.coli BL21(DE3)Gold14pET-20bFrom15forward primer5-GGT TCC GGA TCT GGT TCT GGT TCT CTG GTC CCC Gly Ser Gly Ser Gly Ser Gly Ser Leu Val Pro CGC GGT AGT CAC CAC CAC CAC CAC CAC CGT TTT GTG Arg Gly Ser His His His His His His Arg AAC CAA CAC CTG TGC GGC-3 Proinsulinreverse primer5-AGT GTC GAC TTA GTT GCA GTA GTT CTC CAG CTG GTA-3 Ter ProinsulinTCC GGAGTC GACBspE ISal Isequence of primer16Ajamaluddin Malik, Marco Jenzsch, Andreas Lbbert, Rainer Rudolph,Brigitte ShlingManuscript under revision-Protein Expression and Purification1-21：E. coli ecotin signal sequence22-162： E. coli ecotin 163-174：(Gly Ser)6 linker 175-180: LVPRGS thrombin(凝血酶) cleavage site181-186: (His)6 187: Arg trypsin(胰蛋白酶) cleavage site188-273: ProinsulinSchematic presentation of the ecotin-proinsulin fusion protein cloned in pTrc99a17pCR-Blunt IITOPO18TOPO Cloning Technology19Restriction enzymesBspE I 5 . TC C G G A . 3 3 . A G G C CT . 5 Sal I 5 . GT C G A C . 3 3 . C A G C TG . 5 EcoR I 5 . GA A T T C. 3 3 . C T T A AG . 5 20E.coli BL21(DE3)GoldMore Like a Mammalproduce an increased amount of rare E.coli tRNAs that correspond to codons used more frequently by other organisms.This modification overcomes expression problems due tocodon bias1 .提高提高E.coli tRNAs 结合在其他生物体里广泛使用但在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中很少使用的密码子的机率。的机率。克服了密码子偏爱性造成的表达问题。克服了密码子偏爱性造成的表达问题。E. coli GM2163 (dam- and dcm- ) dam dcm:甲基转移酶甲基转移酶 保护保护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21pEGP1pTrc99acloning vector 2Sequence 4176 BP; 1009 A; 1059 C; 1118 G; 990 T; 0 other; pTrc99A from pTrc98A & pUC18 2pTrc-eco-peps (abbreviated as pEGP1)Express pepsinogen fused to E. coli ecotin11:vorgelegt derA novel strategy for the periplasmic production of heterologous proteins in E. coliT7表达系统表达系统IPTG诱导诱导22Current Protocols in Molecular BiologyJohn Wiley and Sons, Inc.Chapter 1 Escherichia coli, Plasmids, and BacteriophagesSection II Vectors Derived from PlasmidsUnit 1.5 Introduction to Plasmid Biology 23 Construction of pEGP1 plasmid From page23 to page 26 E. coli JM83 The source of ecotin gene3 end of ecotin-(Gly-Ser)3 primerreverse primerPCR pHQPEX-30-515 end (Gly-Ser)3Primerforward primer3 end of pepsinogen(His)6 primerreverse primerPCRforward primer The source of pepsinogenFrom page23 to page26Ajamaluddin Malik, Rainer Rudolph,Brigitte ShlingProtein Expression and PuriWcation 47 (2006) 6626711:Provided by R. Bolli, ZLB Bioplasma AG, Bern,Switzerland24pCR-Blunt II-TOPO kitEcoR I-BspE IBspE I-Sal Ipurified by agarose gel electrophoresis (琼脂糖凝胶电泳琼脂糖凝胶电泳)pTrc99aEcoR I-Sal I dephosphorylatedligate pEGP1pTrc-eco-peps25(A) Structural model of the ecotin dimer.(B) Schematic draw of the ecotin-pepsinogen fusion protein as present in pEGP1.120:ecotin signal peptide21-162:mature ecotin163-174:GS6 linker175-546:mature pepsinogen546-552:His6265-TTC TAA GAA TTC GAA GGA GAT ATA CAT AAT GAA GAC CAT TCT ACC TGC A-3 ecotin signal peptidesequence of primer3 end of ecotin-(Gly-Ser)3 primerreverse primerforword primer5-ATC TTA TCC GGA ACC AGA ACC AGA Gly Ser Gly Ser Gly SerACC ACG AAC TAC CGC ATT GTC AAT TT-3 Gly ecotin5 end (Gly-Ser)3primerforword primer5-GTA TAT GTC GAC TTA GTG GTG GTG GTG Ter His His His HisGTG GTG AGC CAC GGG GGC CAG ACC-3 His His pepsinogen 5-GTT ATA TCC GGA TCT GGT TCT GGT TCT GGT Ser Gly Ser Gly Ser Gly Ser Gly TAC AAG GTC CCC CTC A-3 pepsinogen 3 end of pepsinogen(His)6 primerreverse primerGAA TTCTCC GGAGTC GACBspE IEcoR ISal I27E.coli ecotin gene GENBANK ACCESSION:M60876 J05757 1 cgtagaggat caaaagagta gcgggaagcg tggcaaaaac gggcttttgc tcacatttca 61 aattggttat aaatatattt atatagcgat tgattcacca gagatatttc tgctggtttg 121 ctctctcatt agaatttaac actaaaagag caggtaaaat tgtctgaatg ttctttaagt 181 tattcataaa gcaaattaat aaatctgatg aatatgttaa ccttcagcga catcatcggt 241 gaaaacctat aaatgaagaa ggaaagcaaa aaaatgaaga ccattctacc tgcagtattg 301 tttgccgctt tcgctaccac ttccgcctgg gcggcagaaa gcgtccagcc actggaaaaa 361 atcgcgcctt atccacaagc tgaaaaaggg atgaagcgtc aggtgattca gttaaccccg 421 caagaagatg aatctaccct gaaagtagaa ctgttaatcg gtcagacgct ggaagtcgat 481 tgcaatttgc atcgtctcgg cgggaagctg gaaaacaaaa cgctggaagg ctggggctat 541 gattattatg tctttgataa agtcagttcc ccggtttcaa cgatgatggc ctgcccggat 601 ggcaagaaag agaagaaatt tgtcaccgcg tatctgggcg atgctggaat gctgcgttac 661 aacagcaagc tgccgatcgt ggtgtatacg ccagacaatg tagatgtgaa gtaccgcgtc 721 tggaaggcgg aagagaaaat tgacaacgcg gtagttcgct aaactgccgt gaagtgcggc 781 accccgtagg tcagacaagg cggtcagcga tccgacatcc aacgcccgag ccggttgcct 841 gatgcgacgc tggccgtctt atcaggccta caccgctgtg aagtgcggca ccccgtaggt 901 cagacaaggc ggtcacgcgc atccgacatc caacgcccga gccggttgcc tgatgcgacg 961 ctggcctctt atcaggccta caccgctgtg aagtgctcca ccccgtaggt cggataaggc 1021 ggtagcgatc cgacatctaa cgcaagccgt tgcecotin signal peptidemature ecotin28E.coli ecotin1. Ecotin has no role in the host metabolism. Due to its relatively small size, the metabolic burden is low 1 .2. Prokaryotic signal peptide transport the fusion proteins to the periplasm (周质周质) 2 .3. Play an important role in translocation 2 .4. Ecotin is a broad-range serine-protease inhibitor，when rat trypsinogen was recombinantly produced and secreted into the periplasm of E. coli, it formed a relatively tight and specific complex with E. coli ecotin at neutral pH 1 .5. Due to a strong affinity of ecotin for trypsin as well as trypsinogen，trypsinogen or inactive trypsin variants immobilized to a column can serve as a highly selective affinity material 2 .引导分泌引导分泌防止水解防止水解固定化亲和固定化亲和层析层析 1:Ajamaluddin Malik , Marco Jenzsch , Andreas Lubbert ,Rainer Rudolph , Brigitte Sohling Protein Expression and Purification 55 (2007) 1001112:vorgelegt derA novel strategy for the periplasmic production of heterologous proteins in E. coli代谢负荷低代谢负荷低29References1. Simon Heller , Plamen Kozlovski , Peter Kurtzhals Insulins 85th anniversaryAn enduring medical miracle Diabetes Research and Clinical Practice 78 (2007) 1491582.冯佑民 张友尚 重组人胰岛素研究的回顾和展望 1998, Vol.16, No. 43. Ajamaluddin Malik, Marco Jenzsch, Andreas Lbbert, Rainer Rudolph,Brigitte Shling Periplasmic production of native human proinsulin as a fusion to E. coli ecotin Protein Expression and Purification 55 (2007) 1001114. Ajamaluddin Malik, Marco Jenzsch, Andreas Lbbert, Rainer Rudolph,Brigitte Shling Periplasmic production of native human proinsulin as a fusion to E. coli ecotin Manuscript under revision-Protein Expression and Purification5 .vorgelegt der A novel strategy for the periplasmic production of heterologous proteins in E. coli6. Ajamaluddin Malik, Rainer Rudolph,Brigitte Shling A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm Protein Expression and PuriWcation 47 (2006) 66267130Thank you !