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1、构建胰岛素工程菌构建胰岛素工程菌前言：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 plasmid胰岛素的发现胰岛素的发现1922 Frederick Banting Charles Best人胰岛素一级结构人胰岛素一级结构CHAIN A: 21 amino acidsCHAIN B: 30 amino acids胰
2、岛素立体结构胰岛素立体结构1 翻译后不需要糖基化修饰，在细菌内合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。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，由两条胰岛素是一个相对较小的分子，由两条多肽链组成，一条含多肽链组成，一条含21个氨基酸（个氨基酸（A链）和链）和另一条含另一条含30个氨基酸（个氨基酸（B链）。链）。胰岛素具有两个利于通过胰岛素具有两个利于通过DNA重组技术生产的特重组技术生产的特点：点：重组人胰岛素重组人胰岛素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。1.Humuli
3、n（优泌林）（优泌林） 2.Novolin（诺和灵）（诺和灵） Eli Lilly and Company Novo Nordisk 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 A3.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
4、建重组前胰岛素重组前胰岛素Chain BChain CChain A人胰岛素原表达法基因工程菌的构建战略：tacb-GalC peptideoriAprMetMetMMNCB peptideA peptide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C peptideA peptideB peptideMRRKRCNBrRSSSSCNNCS SS SR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Materials1.Restriction enzymes from NEB and MBI
5、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 fr
6、om 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 Invitro
7、gen14. 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 Prote
8、in Expression and Purification 55 (2007) 100111Construction of the ecotinproinsulin expression plasmid pET 20b-proinsulin The source of proinsulin geneforward primerreverse primerPCRpCR-Blunt II-TOPO kitsequencetransformE. Coli GM2163(dam- and dcm- )pEGP1BspE ISal I(containing the ecotin-pepsinogen
9、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)GoldpET-20bFromforward primer5-GGT TCC GGA TCT GGT TCT GGT TCT CTG GTC CCC Gly Ser Gly Se
10、r 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 primerSchematic presentation
11、of the ecotin-proinsulin fusion protein cloned in pTrc99a1-21：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: ProinsulinpCR-Blunt IITOPORestriction enzymesBspE I 5 .
12、 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 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E.coli BL21(DE3)GoldMore Like a Mammalproduce an increased amount
13、 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中很少使用的密码中很少使用的密码子的机率。子的机率。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。pEGP1pTrc99a2pTrc-eco-peps (abbr
14、eviated 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Current Protocols in Molecular BiologyJohn Wiley and Sons, Inc.Chapter 1 Escherichia coli, Plasmids, and BacteriophagesSecti
15、on II Vectors Derived from PlasmidsUnit 1.5 Introduction to Plasmid Biology Figure 1.5.11 Construction of pEGP1 plasmid From page23 to page 26 E. coli JM83 The source of ecotin genePCR pHQPEX-30-515 end (Gly-Ser)3Primerforward primer3 end of pepsinogen(His)6 primerreverse primerPCRforward primer The
16、 source of pepsinogenpCR-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-peps(A) Structural model of the ecotin dimer.(B) Schematic draw of the ecotin-pepsinogen fusion protein as present in pE
17、GP1.120:ecotin signal peptide21-162:mature ecotin163-174:GS6 linker175-546:mature pepsinogen546-552:His65-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
18、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 TA
19、C AAG GTC CCC CTC A-3 pepsinogen 3 end of pepsinogen(His)6 primerreverse primerGAA TTCTCC GGAGTC GACBspE IEcoR ISal IE.coli ecotin gene GENBANK ACCESSION:M60876 J05757 1 cgtagaggat caaaagagta gcgggaagcg tggcaaaaac gggcttttgc tcacatttca 61 aattggttat aaatatattt atatagcgat tgattcacca gagatatttc tgctgg
20、tttg 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
21、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 tgtcaccgc
22、g 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 aagtgcg
23、gca ccccgtaggt 901 cagacaaggc ggtcacgcgc atccgacatc caacgcccga gccggttgcc tgatgcgacg 961 ctggcctctt atcaggccta caccgctgtg aagtgctcca ccccgtaggt cggataaggc 1021 ggtagcgatc cgacatctaa cgcaagccgt tgcecotin signal peptidemature ecotinE.coli ecotin1. Ecotin has no role in the host metabolism. Due to its
24、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
25、 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 ma
26、terial 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 代谢负荷低代谢负荷低A链和B链分别表达法基因工程菌的构建战略：tactacb-Galb-GalA peptideB peptideorioriAprAprMetMetMetMetMMMM化学合成A链和B链的编码序列A链和B链分别表达法表达产物的后处理路线：MMNCMMNCb-Galb-GalABCNBr 处理NCSSSSCNSSNCNCNCNCCys 体外氧化和重折叠分离纯化A链和B链同时表达法tacb-GaloriAprMetMetMMNCB peptideA peptide化学合成AB链编码序列重组人胰岛素转化分离纯化CNBr 处理特异性裂解体外折叠