pBAD/Thio-TOPO

pBAD/Thio-TOPO

载体基本信息

出品公司:	Ybscience
载体名称:	pBAD/Thio-TOPO
质粒类型:	大肠杆菌表达载体；诱导表达载体
高拷贝/低拷贝:	低拷贝
克隆方法:	TOPO-TA
启动子:	araBAD
载体大小:	4454 bp
5' 测序引物及序列:	Trx Forward: 5′-TTCCTCGACGCTAACCTG-3′
3' 测序引物及序列:	pBAD Reverse 5′-GATTTAATCTGTATCAGG-3′
载体标签:	V5 Epitope（C-端）, 6x His Tag（C-端）; HP-Thioredoxin（N-端）,EK 切割位点
载体抗性:	氨苄青霉素（Ampicillin）
克隆菌株:	TOP10
表达菌株:	推荐LMG194
备注:	pBAD/Thio-TOPO载体是阿拉伯糖调控载体 ,在无葡萄糖的培养基中，阿拉伯糖正向调控目 的基因的表达； 通过调节阿拉伯糖的浓度水平来优化目的蛋白的可溶性表达； 采用TOPO-TA技术，只用5分钟即可将PCR片段连接 到载体上去； pBAD/Thio-TOPO载体表达硫氧还蛋白 (thioredoxin,Trx)融合蛋白，硫氧还蛋白可增加目的蛋白的可溶性，提高蛋白产量。
稳定性:	稳表达
组成型/诱导型:	诱导型（阿拉伯糖)
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

The pBAD/TOPO ThioFusion Expression Kit is designed for one-step cloning and regulated expression of thioredoxin fusion proteins in E. coli(Figure 1). The pBAD/Thio-TOPO vector encodes His-Patch thioredoxin as an N-terminal fusion partner. The thioredoxin fusion can significantly increase the solubility of many difficult-to-express proteins and improve the yield of protein production. 

pBAD/Thio-TOPO 载体具有以下特点：
 The araBAD promoter for tightly regulated expression in E. coli I-activated vector for 5-minute TOPO Cloning of Taq-amplified PCR products
 C-terminal polyhistidine (6xHis) tag for purification with nickel-chelating resin and detection with an Anti-His(C-term) Antibody
 C-terminal V5 epitope for detection with an Anti-V5 Antibody
 Enterokinase cleavage site for efficient cleavage of N-terminal fusion tag with EKMax Enterokinase

pBAD/TOPO ThioFusion Expression Kit provides a highly efficient, 5-minute, one-step cloning strategy ("TOPO Cloning") for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector for soluble, regulated expression and simplified protein purification in E. coli. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required. Expression in E. coli is driven by the araBAD promoter (PBAD). The AraC gene product encoded on the pBAD/Thio-TOPO plasmid positively regulates this promoter. Recombinant proteins are expressed as fusions to His-Patch thioredoxin for high-level expression and simple purification.

L-阿拉伯糖调控表达
In the presence of arabinose, expression from PBAD is induced while only very low levels of transcription are observed from PBAD in the absence of arabinose (Lee, 1980; Lee et al., 1987). Uninduced levels are repressed even further by growth in the presence of glucose (0.1% to 0.2%). Glucose reduces the levels of 3′, 5′-cyclic AMP, lowering expression from the catabolite-repressed PBAD promoter (Miyada et al., 1984). By varying the concentration of arabinose, protein expression levels can be optimized to ensure maximum expression of protein. In addition, the tight regulation of PBAD by AraC is useful for expression of potentially toxic or essential genes (Carson et al., 1991; Dalbey and Wickner, 1985; Guzman et al., 1992; Kuhn and Wickner, 1985; Russell et al., 1989; San Millan et al., 1989). 

硫氧还蛋白
The 11.7 kDa thioredoxin protein is found in yeast, plants, and mammals, as well as in bacteria. It was originally isolated from E. coli as a hydrogen donor for ribonuclease reductase (for a review, see Holmgren, 1985 ). The gene has been completely sequenced (Wallace and Kushner, 1984). The protein has been crystallized and its three-dimensional structure determined (Katti et al., 1990).
When overexpressed in E. coli, thioredoxin is able to accumulate to approximately 40% of the total cellular protein and still remains soluble. Thioredoxin is used to increase translation efficiency, and in some cases, solubility, of eukaryotic proteins expressed in E. coli. Murine interleukin-2, human interleukin-3, murine interleukin-4, murine interleukin-5, human macrophage-colony stimulating factor, murine steel factor, murine leukemia inhibitory factor and human bone morphogenetic protein-2 are some of the proteins that have been produced as soluble C-terminal fusions to the thioredoxin protein in E. coli (LaVallie et al., 1993).

带有组氨酸补丁的硫氧还蛋白
To create a metal binding domain in the thioredoxin protein, the glutamate residue at position 32 and the glutamine residue at position 64 were mutated to create histidine residues. When His-Patch thioredoxin folds, the histidines at positions 32 and 64 interact with a native histidine at position 8 to form a "patch". This histidine patch was shown to have high affinity for divalent cations (Lu et al., 1996). His-Patch thioredoxin (HP-thioredoxin) proteins can therefore be purified on metal-chelating resins (e.g., ProBond )

 

载体序列

pBAD/Thio-TOPO  4454BP

AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAAC
CAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGT
AACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATG
CCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTC
TCCATACCCGTTTTTTTGGGCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATACCCATGGG
ATCTGATAAAATTATTCATCTGACTGATGATTCTTTTGATACTGATGTACTTAAGGCAGATGGTGCAATC
CTGGTTGATTTCTGGGCACACTGGTGCGGTCCGTGCAAAATGATCGCTCCGATTCTGGATGAAATCGCTG
ACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCACAACCCGGGCACTGCGCCGAAATA
TGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCA
CTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGCTCTGGATCCGGTGATGACGATG
ACAAGCTCGCCCTTAAGGGCGAGCTTGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTAC
GCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAG
AGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGG
CGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGT
AGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA
GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAG
CGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCA
TCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTT
TCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA
AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT
GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT
ACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT
GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATG
GCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT
GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAA
TGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA
CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT
GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT
CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA
GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT
TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCC
CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC
TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG
GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCC
TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT
AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG
TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA
CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGC
GGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC
TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAG
GGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTT
TGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT
GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGA
TGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTG
CTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCC
GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGC
TGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAG
ATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGC
AAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTAC
ATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACC
CGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGC
TCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTG
GCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTG
CTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCG
ACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATA
GCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCA
TCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCG
GACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCC
TGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGA
CCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAAC
CGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATC
ATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAG