Bacterial Expression vector

• Introduction

• Expression of cloned genes in Escherichia coli

• Expression of cloned genes in other bacteria

• Conclusion

Introduction

promoter

plasmid copy number

activity of protein (fusion protein)

Introduction

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

promoter

bacteriophage PLpromoter 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.

Lac promoter

Lac promoter

Lac promoter

trp promoter

trp promoter

plasmid copy number

pET vectorpET vector

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

Fusion protein

Table 15-1 Vectors for Construction of Fusion Proteins

Fusion protein

Vector System Fusion Partnera Comment Reference/Source

pUC, pSK,pBluescript, pGEM

-galactosidase Expression under control of the lac promoter-operator system.

Stratagene (www.stratagene.com)Life Technologies (www.lifetech.com)

pTA1529 or pBAce alkaline phosphatase

PhoA signal sequence facilitates transport to periplasm.

Oka et al.

pGEX series GST IPTG-inducible promoter; available with cleavage sequences.

Smith and Johnson (1988)Pharmacia

pMAL series MBP IPTG-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, pTrxFus Trx IPTG-inducible promoter; available with enterokinase cleavage sequences.

La Vallie et al. (1993)Invitrogen (www.invitrogen.com)

pET series poly-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)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

Fusion protein

Fusion protein 的切除

化學法 (chemical)

酵素法 (enzyme)

Affinity purification

Fusion protein

Fusion protein 的去除：酵素法

Fusion protein

Fusion protein 的去除： affinity purification

Expression of cloned genes in E. coli

pGEX system

pET system

pQE system

pGEX system

promoter: Ptac (trp-lac promoter)

fusion protein: GST (glutathione S-transferase)

pGEX system

pGEX system

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

pET system

pET system

Stratagene

pET3 系列 : promoter 為 T7

Stratagene

pET11 系列 : promoter 為 T7/lac O

Novagen