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1、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:Construction of the ecotin(大肠杆菌素)proinsulin expression plasmidConstruction of pEGP1 plasmid,胰岛素的发现,1922 Frederick Banting Charles Best,人胰岛素一级结构,CHAIN A:21 amino acidsCHAIN B:30 amino acids,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
2、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),Eli Lilly and Company,Novo Nordisk,欧洲市场的重组胰岛素类似物,重组人胰岛素基因工程菌的构建,人胰岛素原表达法 获取人胰岛素cDNA,克隆在含有Ptac和-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Ar
3、g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/g,Materials,1.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-
4、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,Cop
5、enhagen.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
6、15 Ajamaluddin Malik,Marco Jenzsch,Andreas Lubbert,Rainer Rudolph,Brigitte Sohling Protein Expression and Purification 55(2007)100111,pET-20b,From,Schematic presentation of the ecotin-proinsulin fusion protein cloned in pTrc99a,1-21:E.coli ecotin signal sequence22-162:E.coli ecotin 163-174:(Gly Ser)
7、6 linker 175-180:LVPRGS thrombin(凝血酶)cleavage site181-186:(His)6 187:Arg trypsin(胰蛋白酶)cleavage site188-273:Proinsulin,pCR-Blunt IITOPO,E.coli GM2163(dam-and dcm-)dam dcm: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.coli BL21(DE3)GoldMore Like a Mammalproduce an increased amount of rare E.coli tRNAs that
8、 correspond to codons used more frequently by other organisms.This modification overcomes expression problems due tocodon bias1.提高E.coli tRNAs 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。,Current Protocols in Molecular BiologyJohn Wiley and Sons,Inc.Chapter 1 Escherichia coli,Plasmids,and Bac
9、teriophagesSection II Vectors Derived from PlasmidsUnit 1.5 Introduction to Plasmid Biology Figure 1.5.11,pCR-Blunt II-TOPO kit,EcoR I-BspE I,BspE I-Sal I,purified by agarose gel electrophoresis(琼脂糖凝胶电泳),pTrc99a,EcoR I-Sal I,dephosphorylated,ligate,pEGP1pTrc-eco-peps,E.coli ecotin gene GENBANK ACCES
10、SION: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
11、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 atcgtctcg
12、g 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 gtagttc
13、gct 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 tgc,AB链分别表达法 化学合成A链和B链的编码区 分别克隆在含Ptac和-gal的表达型质粒上,连接点上为Met的密码子 分别表达出融合蛋白,通过CNBr处理,纯化A链和B链 A链和B链按2:1比例混合,体外进行化学折叠结果:由于胰岛素上有3各二硫键,其正确配对率仅1020,成本为$180/g,