The pBAD-TOPO TA Expression Kit is specifically designed for one-step cloning and regulated prokaryotic expression of Taq-amplified PCR products. Once you've TOPO Cloned your PCR product, you can go straight to protein expression. Some of the convenient features of the pBAD-TOPO vector include:
Linearized, topoisomerase I-activated vector for 5-minute cloning of Taqamplified PCR products
The araBAD promoter for tightly regulated expression in E. coli V5 epitope tag for detection with an Anti-V5 Antibody
C-terminal polyhistidine (6xHis) tag for purification using nickel-chelating resin and detection with an Anti-His(C-term) Antibody
Enterokinase cleavage site for removal of N-terminal leader peptide
pBAD-TOPO TA Expression Kit provides a highly efficient, 5-minute, one-step cloning strategy ("TOPO Cloning") for the direct insertion of Taq polymeraseamplified PCR products into a plasmid vector for regulated expression 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-TOPO plasmid positively regulates this promoter. L-阿拉伯糖调控表达 In the presence of L-arabinose, expression from PBAD is turned on while the absence of L-arabinose produces very low levels of transcription from PBAD (Lee, 1980; Lee et al., 1987). Uninduced levels are repressed even further by growth in the presence of glucose. Glucose reduces the levels of 3′, 5′-cyclic AMP, thus lowering expression from the catabolite-repressed PBAD promoter (Miyada et al., 1984). By varying the concentration of L-arabinose, protein expression levels can be optimized to ensure maximum expression of soluble 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). For more information on the mechanism of expression and repression of the ara regulon. TOPO克隆技术 The PCR expression vector (pBAD-TOPO) is supplied linearized with:
Single 3′-thymidine (T) overhangs for TA Cloning
Topoisomerase (bound to the vector)
Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3′ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3 phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5 hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994).
