1. Bacterial Expression vector Introduction Expression of cloned genes in Escherichia coli Expression of cloned genes in other bacteria Conclusion

2. Introduction  promoter  plasmid copy number  activity of protein (fusion protein)

3. Introduction

4. promoter  可誘導式 (inducible) 適用於希望將宿主細胞的生長階段 (growth phase) 與蛋白 質的生產階段 (production phase) 加以區分，同時將生產 蛋白質的時間儘量縮短，使醱酵培養得以順利進行  非誘導式 (non-inducible ； constitutive) 適用於基因工程蛋白質對宿主細胞的生長沒有影響 Introduction

5. promoter

6.  bacteriophage P L promoter regulated by a temperature-sensitive repressor cIts857, which represses transcription at low temperature tac promoter (trp-lac promoter) -35 region of the trp promoter -10 region of the lac UV5 promoter trc promoter (trp-lac promoter) -35 region of the trp promoter -10 region of the lac UV5 promoter 16 bp. 17 bp.

7. Lac promoter

10. trp promoter

12. plasmid copy number pET vector

13. Fusion protein 利於分離目標蛋白質 有些 carrier protein 可以保護目標蛋白質免受蛋白質 水解酵素的破壞 有些 carrier protein 可以幫助目標蛋白質增加溶解度 避免形成不可溶的 inclusion bodies

14. Fusion protein

15. Table 15-1 Vectors for Construction of Fusion Proteins Fusion protein Vector SystemFusion Partner a CommentReference/Source pUC, pSK, pBluescript, pGEM  -galactosidase Expression under control of the lac promoter-operator system. Stratagene (www.stratagene.com)www.stratagene.com Life Technologies (www.lifetech.com) pTA1529 or pBAcealkaline phosphatase PhoA signal sequence facilitates transport to periplasm. Oka et al. pGEX seriesGSTIPTG-inducible promoter; available with cleavage sequences. Smith and Johnson (1988) Pharmacia pMAL seriesMBPIPTG-inducible promoter; MBP signal sequence facilitates export to periplasm. di Guan et al. (1988); Maina et al.(1988) New England Biolabs (www.neb.com) pTrx, pTrxFusTrxIPTG-inducible promoter; available with enterokinase cleavage sequences. La Vallie et al. (1993) Invitrogen (www.invitrogen.com)www.invitrogen.com pET seriespoly-His tag, selected vectors also carry tags for GST, Trx, DsbA, and DsbC, CBD T7 promoter (IPTG-inducible); available with sites for chemical, cleavage. Studier et al. (1990) Novagen (www.novagen.com)www.novagen.com Promega (www.promega.com) a (GST) Glutathione S-transferase; (MBP) maltose-binding protein; (Trx) thioredoxin; (DsbA and Dsbc) disulfide bond formation (periplasmic localization); (CBD) cellulose-binding domain. 大部分的 fusion protein 均可以 affinity chromatography 純化出來 目前約有 20 種不同的 fusion protein system

16. Fusion protein Fusion protein 的切除 化學法 (chemical) 酵素法 (enzyme) Affinity purification

17. Fusion protein

18. Fusion protein 的去除：酵素法

19. Fusion protein

20. Fusion protein 的去除： affinity purification

26. Expression of cloned genes in E. coli  pGEX system  pET system  pQE system

27. pGEX system promoter: Ptac (trp-lac promoter) fusion protein: GST (glutathione S-transferase)

28. pGEX system

32. pET system promoter: T7 promoter / T7 promoter + lac operon fusion protein: poly-His tag, GST, MBP, Trx, DsbA and DsbC,CBD (GST) Glutathione S-transferase; (MBP) maltose-binding protein; (Trx) thioredoxin; (DsbA and Dsbc) disulfide bond formation (periplasmic localization); (CBD) cellulose-binding domain

33. pET system

35. Stratagene pET3 系列 : promoter 為 T7

36. Stratagene pET11 系列 : promoter 為 T7/lac O

37. Novagen