| 出品公司: | Invitrogen |
|---|---|
| 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。 |
| 产品目录号: | K370-01 |
| 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 |
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pBAD/Thio-TOPO载体质粒基本信息
pBAD/Thio-TOPO质粒图谱载体图谱和pBAD/Thio-TOPO载体序列质粒序列多克隆位点信息
pBAD/Thio-TOPO质粒载体简介
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 )
pBAD/Thio-TOPO质粒序列载体序列
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
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pBAD/Thio-TOPO质粒载体应用举例
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