pBAD/Thio-TOPO

pBAD/Thio-TOPO

pBAD/Thio-TOPO载体基本信息

pBAD/Thio-TOPO载体质粒图谱和多克隆位点信息

pBAD/Thio-TOPO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：

Þ      The araBAD promoter for tightly regulated expression in E. coli I-activated vector for 5-minute TOPO Cloning of Taq-amplified PCR products

Þ      C-terminal polyhistidine (6xHis) tag for purification with nickel-chelating resin and detection with an Anti-His(C-term) Antibody

Þ      C-terminal V5 epitope for detection with an Anti-V5 Antibody

Þ      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

pBAD/Thio-TOPO其他大肠杆菌表达载体：