Upload
beverly-lucas
View
223
Download
7
Embed Size (px)
Citation preview
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