E. COLI EXPRESSION VECTORS
pALEXa, b and c
pALEXa, b and c vectors contain the 3´ moiety of the Streptococcus pneumoniae lytA gene (C-LYTAG protein) between the Pm promoter/lac operator and the multiple cloning site. This vectors allows the expression of fusion protein to C-LYTAG with CASCADE ™ system.
pALEX vectors contain a multiple cloning site (MCS) that has seven unique restriction sites: BamHI, XhoI, SacI, BglII, KpnI, BstBI and HinDIII (refer to vector map for details). They are available in all three reading frames (pALEXa, pALEXb and pALEXc), to facilitate the traductional fusion of the interest gene to the codificant region of C-LYTAG.
pALEX vectors carry as selection marker the bla gene, that confers ampicillin resistance and include an enterokinase recognition sequence that enables removal of the C-LYTAG moiety from the fusion protein.
pALEX1a, b and c
pALEX1a, b and c are highly improved CASCADE ™ bacterial expression vectors to be use in E. coli hosts strains bearing the salicylate-inducible nahR/Psal::xylS regulatory module, such as REG1 or REG-12.
These new vectors are high-copy number, ampicilin-selectable plasmids that incorporate a mutant Pm promoter with even lower basal transcriptional activity, for tightly regulated and high capacity protein expression. Among other features, pALEX1a, b and c have an improved multiple cloning site (MCS) with target for 20 different restriction enzymes, for easy and flexible cloning in-frame with an ATG start codon located at an optimal distance from a highly efficient ribosome binding site (RBSII), as well as stop codons in all three forward reading frames, followed by strong transcriptional terminator sequences (t0 and T1).
The expression cassette in pALEX1a, b and c is flanked by “rare cutter” NotI sites, for convenient subcloning into mini Tn5 delivery vectors and construction of genetically stable expression strains, its stable insertion in the bacterial chromosome
pALEX2a, b and c
The new pALEX2 vector series is designed to be used with the CASCADE ™ system for the expression, purification and immobilization of proteins fused in their N-termini to LYTAG, using the LYTRAP chromatographic resin and its related reagents. The new tag (136 aa, 15.21 KDa) incorporates an alpha helix polypeptidic sequence which acts as a spacing element between domain to choline union and the interest protein, reducing the potential steric interference between both regions and facilitating, if necessary, the elimination of the tag by enterokinase digestion
The pALEX2 vectors are available in three reading frames (pALEX2a, pALEX2b and pALEX2c). The pALEX2 are derivatives from pALEX1 (pALEX1a, pALEX1b and pALEX1c) and therefore have a multiple cloning site, another interesting feature is the presence of a unique restriction site for the blunt cutter PshAI overlapping with the sequence encoding the enterokinase recognition site, allowing its direct fusion with the protein of interest and recovery of a product without undesired N-terminal amino acid residues, after enterokinase cleavage. The pALEX2 vectors carry as selection marker the bla gene that confers ampicillin resistance.
The expression cassette in pALEX2a, b and c is flanked by “rare cutter” NotI sites, for convenient subcloning into mini Tn5 delivery vectors and construction of genetically stable expression strains for its stable insertion in the bacterial chromosome.
pCNB4-S2 is a mini-Tn5 Km transposon delivery plasmid designed for stable integration of the CASCADE ™ regulatory module in the chromosome of a wide variety of Gram-negative bacteria. pCNB4-S2 delivers a mobile element expressing xylS under the control of the Psal promoter and its cognate salicylate-responsive regulator NahR. Transposition of the CASCADE ™ regulatory module from this plasmid depends on the expression of a transposase gene in pCNB4-S2 external to the mobile element, allowing stability of the transposed module by simply curing of pCNB4-S2 from the recipient bacterial strain.
pCCD5 is a 20-40 copy number, 3.4 Kb, ColE1-derived vector usable for cloning and expression of protein genes in E. coli with the CASCADE™ system. pCCD5 contains the Pm promoter preceded by a transcriptional terminator, and located upstream of a multi-cloning site that includes an efficient translation initiation region.