構(gòu)建胰島素基因工程菌（課堂PPT）

1、.1構(gòu)建胰島素工程菌構(gòu)建胰島素工程菌.2前言：前言：胰島素的發(fā)現(xiàn)胰島素的發(fā)現(xiàn)胰島素的結(jié)構(gòu)胰島素的結(jié)構(gòu)重組人胰島素及類型重組人胰島素及類型重組人胰島素基因工程菌的構(gòu)建：重組人胰島素基因工程菌的構(gòu)建：Construction of the ecotin(大腸桿菌素大腸桿菌素)proinsulin expression plasmidConstruction of pEGP1 plasmid.3胰島素的發(fā)現(xiàn)胰島素的發(fā)現(xiàn)1922 Frederick Banting Charles Best.4人胰島素一級(jí)結(jié)構(gòu)人胰島素一級(jí)結(jié)構(gòu)CHAIN A: 21 amino acidsCHAIN B: 30 amin

2、o acids.5胰島素立體結(jié)構(gòu)胰島素立體結(jié)構(gòu).61 翻譯后不需要糖基化修飾，在細(xì)菌內(nèi)合翻譯后不需要糖基化修飾，在細(xì)菌內(nèi)合成的胰島素具有完整的生物活性。成的胰島素具有完整的生物活性。2 胰島素是一個(gè)相對(duì)較小的分子，由兩條胰島素是一個(gè)相對(duì)較小的分子，由兩條多肽鏈組成，一條含多肽鏈組成，一條含21個(gè)氨基酸（個(gè)氨基酸（A鏈）鏈）和另一條含和另一條含30個(gè)氨基酸（個(gè)氨基酸（B鏈）。鏈）。胰島素具有兩個(gè)利于通過(guò)胰島素具有兩個(gè)利于通過(guò)DNA重組技術(shù)生產(chǎn)的特重組技術(shù)生產(chǎn)的特點(diǎn)：點(diǎn)：.7重組人胰島素重組人胰島素1982年年10月在美國(guó)上市，全球第一月在美國(guó)上市，全球第一個(gè)商業(yè)化基因重組人胰島素。個(gè)商業(yè)化基因重

3、組人胰島素。1.Humulin（優(yōu)泌林）（優(yōu)泌林） 2.Novolin（諾和靈）（諾和靈） Eli Lilly and Company Novo Nordisk .8歐洲市場(chǎng)的重組胰島素類似物歐洲市場(chǎng)的重組胰島素類似物.9重組人胰島素類型重組人胰島素類型1.重組前胰島素重組前胰島素Chain BChain CChain A2.A,B鏈分開(kāi)表達(dá)鏈分開(kāi)表達(dá) +Chain BChain A.103.B-miniC-A 重組表達(dá)重組表達(dá)Chain BChain AMini Chain C4.B-XXX-A 重組表達(dá)重組表達(dá) X為氨基酸為氨基酸 Chain BChain AX1X2Xn N3.11重組人

4、胰島素基因工程菌的構(gòu)建重組人胰島素基因工程菌的構(gòu)建重組前胰島素重組前胰島素Chain BChain CChain A.12人胰島素原表達(dá)法基因工程菌的構(gòu)建戰(zhàn)略：tacb-GalC peptideoriAprMetMetMMNCB peptideA peptide人胰島素原的cDNA重組人胰島素原轉(zhuǎn)化分離純化.13人胰島素原表達(dá)法表達(dá)產(chǎn)物的后處理路線：SSSSCN胰蛋白酶C peptideA peptideB peptideMRRKRCNBrRSSSSCNNCS SS SR羧肽酶BB鏈中第22位上的Arg和第29位上的Lys由于良好折疊的原因，對(duì)胰蛋白酶不敏感.14.15Materials1.R

5、estriction 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 laboratory

6、7.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 Peq

7、lab13. 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 ,Rai

8、ner Rudolph , Brigitte Sohling Protein Expression and Purification 55 (2007) 100111.16Construction of the ecotinproinsulin expression plasmid pET 20b-proinsulin The source of proinsulin geneforward primerreverse primerPCRpCR-Blunt II-TOPO kitsequencetransformE. Coli GM2163(dam- and dcm- ).17pEGP1Bsp

9、E 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)Gold.18pET-20bFrom.19forward primer5-GGT TCC GG

10、A 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

11、 ISal Isequence of primer.20Schematic presentation 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: Proi

12、nsulin.21pCR-Blunt IITOPO.22.23Restriction 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 .24E. coli GM2163 (dam- and dcm- ) dam dcm:甲基轉(zhuǎn)移酶甲基轉(zhuǎn)移酶 保護(hù)保護(hù)DNA序列不被甲基化，提高限制性內(nèi)切酶正確識(shí)別酶切位點(diǎn)的機(jī)率序列不被甲基化，提高限制性內(nèi)切酶正確識(shí)別酶切位點(diǎn)的機(jī)率E.

13、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 結(jié)合在其他生物體里廣泛使用但在結(jié)合在其他生物體里廣泛使用但在E.coli中很少使用的密碼中很少使用的密碼子的機(jī)率。子的機(jī)率?？朔?/p>

14、密碼子偏愛(ài)性造成的表達(dá)問(wèn)題?？朔嗣艽a子偏愛(ài)性造成的表達(dá)問(wèn)題。.25pEGP1pTrc99a2pTrc-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表達(dá)系統(tǒng)表達(dá)系統(tǒng)IPTG誘導(dǎo)誘導(dǎo).26Current Protocols in Molecular BiologyJohn Wiley and So

15、ns, Inc.Chapter 1 Escherichia coli, Plasmids, and BacteriophagesSection II Vectors Derived from PlasmidsUnit 1.5 Introduction to Plasmid Biology Figure 1.5.11.27 Construction of pEGP1 plasmid From page23 to page 26 E. coli JM83 The source of ecotin genePCR pHQPEX-30-515 end (Gly-Ser)3Primerforward p

16、rimer3 end of pepsinogen(His)6 primerreverse primerPCRforward primer The source of pepsinogen.28pCR-Blunt II-TOPO kitEcoR I-BspE IBspE I-Sal Ipurified by agarose gel electrophoresis (瓊脂糖凝膠電泳瓊脂糖凝膠電泳)pTrc99aEcoR I-Sal I dephosphorylatedligate pEGP1pTrc-eco-peps.29(A) Structural model of the ecotin dim

17、er.(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:His6.305-TTC TAA GAA TTC GAA GGA GAT ATA CAT AAT GAA GAC CAT TCT ACC TGC A-3 ecotin signal peptidesequence of primer3 end

18、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 pepsin

19、ogen 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 I.31E.coli ecotin gene GENBANK ACCESSION:M60876 J05757 1 cgtagaggat caaaagagta gcgggaagcg tggcaaaaac gg

20、gcttttgc 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 3

21、01 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 tctttg

22、ataa 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 tccg

23、acatcc 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 ecotin.32E.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 (周質(zhì)周質(zhì)) 2 .