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Recombinant DNA Technology

Recombinant DNA Technology. Dr. Hui LI Office : S408 Tel: 26538722. Topic 5 Expression of recombinant gene (Prokaryotes). 1. Gene expression. 遗传信息从 DNA 到蛋白质的传递过程 —— 中心法则( central dogma )。. 2. Expression of recombinant gene. 3. Expression of recombinant gene in prokaryote.

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Recombinant DNA Technology

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  1. Recombinant DNA Technology Dr. Hui LI Office : S408 Tel: 26538722

  2. Topic 5 Expression of recombinant gene (Prokaryotes)

  3. 1. Gene expression 遗传信息从DNA到蛋白质的传递过程——中心法则(central dogma)。

  4. 2. Expression of recombinant gene

  5. 3. Expression of recombinant gene in prokaryote Prokaryotic promoter SDsequence MCS terminator A dividing E. coli

  6. 3. 1 Remarks on prokaryotic expression system 1. 只有一种RNA多聚酶 识别原核细胞的启动子,催化所有的RNA合成。 2. 以操纵子(operon)为单位 数个相关的结构基因与其调控区结合形成一个表达的协同单位。

  7. 3. 转录和翻译偶联、连续进行。 5’ 3’ 有意义链 3’ 5’ 反意义链 转录 5’ 3’ mRNA 翻译 N C 蛋白质

  8. Shine-Dalgarno(S-D)sequence: 4. 不含内含子(intron),缺乏转录后的加工系统。 5. 调控主要在转录水平上。 6. mRNA的核糖体结合位点。 含有一个启始密码子和一段同核糖体16SRNA3’末端碱基互补的序列,叫Shine-Dalgarno(S-D)序列。

  9. 3.2 Attention when we express recombinant gene in prokaryotes 1. 外源基因不能带有内含子。 2. 必须用cDNA 3. 不能直接用真核基因组DNA。 4. 必须利用原核细胞的调控原件(启动子等) 5. 防止外源基因产物对宿主细胞的毒害。

  10. 3.3 Principal factors in bacterial expression

  11. 3.3 Regulation of prokaryotic gene expression 1. 启动子 (promoter) 是DNA上的能与RNA聚合酶结合并能起始mRNA合成的序列。 (1) 启动子序列 大肠杆菌的所有启动子中都有两段一致顺序(consensus sequence)。 -35 Box and -10 Box

  12. E. coli Promoter Sites

  13. consensus sequences

  14. ① -35box RNA聚合酶 亚基的识别位点 。 5’-TTGACA-3’ ② -10box(Pribnow Box): The Pribnow box has a function similar to the TATA box that occurs in promoters in eukaryotes and archaea: it is recognized and bound by a subunit of RNA polymerase during initiation of transcription 5’-TATAAT-3’ 5’ TATAAT 转录起始位点 TTGACA 17bp 核糖体结合位点

  15. 结合区

  16. (2)翻译的起始位点 ①核糖体结合位点( RBS) ribosome binding site i)Shine-Dalgarno(SD) sequence: mRNA上与核糖体16sRNA结合的序列。 SD mRNA 5’—AGGAGGU——AUG—— UCCUCCA 16S rRNA3’ S-D序列距离AUG的距离也影响翻译

  17. ii)起始密码: 位于SD序列下游。 AUG(91%) GUG(8%) UUG(1%)

  18. 2. RNApolymerase 大肠杆菌只有一种类型的RNA多聚酶转录tRNA,rRNA和mRNA。 (1)结构 全酶是一个5聚体,含有两个α小亚基,和2两个大亚基(β和β′),一个σ亚基。

  19. 3. 转录终止子 在表达载体克隆位点的下游一般设计一段转录终止子。 启动子 操纵子 S-D序列 目的基因 终止子 内终止子 intrinsic terminator: E.coli中促使转录终止的DNA位置有一段反向回文顺序,其后紧接一串A,称为内终止子,形成终止信号。

  20. 原因: ① 茎环结构 反向回文顺序被转录后,立即形成一个茎环结构,使转录物与模板之间配对的碱基数降低,整个减弱了RNA 与DNA的互作用 ② 多聚A/U 由于茎环3’段紧接一串A/U的配对,稳定性比较差,有利于转录物脱落而不利于转录延续。

  21. 5-CCCACAGCCGCCAGTTCCGCTGGCGGCATTTTAA CT TCT TTCT-3 3-GGGTGTCGGCGGTCAAGGCGACCGCCGTAAAATTGAAGAAAGA-5(模板) 转录↓ 5-CCCACAGCCGCCAGUUCCGCUGGCGGCAUUUU-OH-3(RNA) RNA折叠↓ C U C U G 脱落 G—C A—U mRNA折叠 C—G C—G G—C C—G RNA聚合酶 C—G G—C A A CCCAC UUUU—3 DNA

  22. 4. 翻译终止密码 (Stop codon) 大肠杆菌偏爱UAAU。一般安置上全部的三个终止密码防止核糖体跳跃(skipping)。 5. 翻译增强子(Enhancer) 能够显著增强外源基因在大肠杆菌细胞中的表达效率的特殊序列。

  23. 6. 基因工程常用的原核启动子 (1)最佳启动子必须具备的条件 ① 必须是一种强启动子 能使外源基因的蛋白产量达到细胞总蛋白的10%-30%以上。 ② 应呈现低水平的基础转录 便于表达毒性蛋白等。 ③应是可诱导型的 用温度或化学试剂诱导。

  24. Inducible bacterial promoters Why not to use constitutive, always strong promoter? Bacterial grow takes time…. Because recombinant (alien) protein is often toxic for bacterial cell. Bacteria tend to expel harmful plasmids Induction

  25. (2) 乳糖启动子lac 来自大肠杆菌的乳糖操纵子。 用乳糖或其类似物IPTG充当诱导物,与阻遏蛋白结合,解除抑制。 Plac O 目的基因

  26. 阻遏物与操纵基因结合

  27. 四聚体的阻遏物

  28. 阻遏物与DNA的结合

  29. ①乳糖操纵子控制区的结构 “可移动的lac启动子小片断” 组成: 阻遏物作用区 CAP作用区 RNA聚合酶作用区 长度: 203bp的HaeIII片断 (包括β-半乳糖苷酶的前8个密码)。

  30. IPTG =异丙基硫代-β-D半乳糖 IPTG有毒、昂贵,不理想。

  31. BL(DE3) inducible system and pET vectors (invented in 1984 by Bill Studier, on sale by Novagen) Gene of interest is expressed from strong T7 promoter pET23 1) T7 RNA polymerase gene is integrated in chromosome under the control of a lac promoter and operator 2) lactose analogue, IPTG, causes the host to produce T7 RNA polymerase • 3) The E. coli host genome also carries the lacI (repressor) gene

  32. Why repressor gene and gene of interest are expressed from different DNA molecules? Repressor gene expressed from chromosome; Gene of Interest expressed from plasmid If too high repressor no transcription (you need to increase expensive IPTG) If too low repressor promoter is leaky (active without IPTG) Repressor is in chromosome, because there it is best kept controlled there (no plasmid loss, not too high expression)

  33. (3)色氨酸启动子trp 来自大肠杆菌的色氨酸操纵子。 trpR P1 O  trpE trpD P2 trpC trpB trpA P1 启动子; trpR 阻遏蛋白基因; P2 衰减子  操纵基因; O

  34. 色氨酸操纵子

  35. (P1是主要启动子,P2的作用只有3%。) 没有色氨酸时,阻遏蛋白不能与操纵基因结合,能转录合成色氨酸所需要的酶。 trpR P1 O  trpE trpD P2 trpC trpB trpA 链 链 转录 阻遏物 邻氨基苯甲 酸合成酶 色氨酸 合成酶 吲哚甘油 硼酸合成酶 邻氨基 苯甲酸 磷酸核糖基 邻氨基苯甲酸 分支酸 吲哚甘 油-磷酸 CDRP 色氨酸

  36. 有色氨酸时,阻遏蛋白与色氨酸结合后才能与操纵基因结合,从而阻止色氨酸合成酶类的转录(称为corepressor)。有色氨酸时,阻遏蛋白与色氨酸结合后才能与操纵基因结合,从而阻止色氨酸合成酶类的转录(称为corepressor)。 trpR P1 O  trpE trpD P2 trpC trpB trpA 结合 不转录 阻遏物 色氨酸 用于表达载体的trp启动子一般只包含启动基因、操纵基因、和部分trpE基因。 P1 O trpE 目的基因

  37. (4)PL和PR启动子 是从噬菌体中得到的一类启动子,比lac启动子的活性高8-10倍,比trp启动子活性高。 cIII N PL/OL cI PM OR/PR Cro PE cII N:抗终止蛋白; Cro: 抗阻遏蛋白(裂解必需的); cI, cII, cIII: 阻遏蛋白; P:启动子;O:操纵子。 R:右;L:左

  38. 转录 早期左边 转录 cIII N PL/OL cI PM OR/PR Cro PE cII 早期右边 阻遏物 转录 当cI合成后,与OL和OR结合阻止RNA聚合酶,则左右基因都受到抑制。但促进PM使cI进一步转录。进入溶原期。

  39. cI857: 表达载体常用的噬菌体启动子是PL。 调节基因cI 则是选择了一个温度敏感性的突变基因cI857。 整合在宿主基因组里,或克隆到载体上。 cI857 PL 外源基因 28oC时阻遏PL,外源基因不转录。 42oC时阻遏蛋白失活,外源基因大量转录

  40. 3.4 Where your expressed protein will be located? Secreted (!!) E.Coli can not do that Inclusion bodies (insoluble) Cytoplasm (soluble) Periplasmatic space (soluble or insoluble)

  41. Inclusion bodies (most common case) -- Inclusion bodies are formed through the accumulation of folding intermediates rather than from the native or unfolded proteins. -- It is not possible to predict which proteins will be produced as inclusion bodies. -- Production of inclusion bodies not dependent on the origin of protein, the used promoters, the hydrophobicity of target proteins...

  42. Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell Protein Folding Page 116

  43. Good side of inclusion bodies • inclusion bodies can be accumulated in the cytoplasm • to much higher level (greater than 25%) • than production as soluble form; 2) inclusion bodies is initially isolated in a highly purified, solid, and concentrated state by simple physical operation (centrifugation). 3) inclusion bodies have no biological activity. For toxic proteins it may be the only one available; 4) inclusion bodies areresistant to proteolysis That results in the high yield of protein production.

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