PROTEIN EXPRESION AND PURIFICATION - Expresion Systems - Vectors

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pCNB4-S2

pCNB4-S2 is a mini-Tn5 Km transposon delivery plasmid designed for stable integration of the CASCADETM 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 CascadeTM 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.

REF.	PRODUCT	FORMAT
EV-3407	pCNB4-S2	8 µg

pCCD5

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 CASCADETM 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.

REF.	PRODUCT	FORMAT
EV-3408	pCCD5	8 µg

pALEXa, b and c vectors

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 CASCADETM 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.

REF.	PRODUCT	FORMAT
EV-3240	pALEX-a	8 µg
EV-3241	pALEX-b	8 µg
EV-3242	pALEX-c	8 µg
EV-3413	pALEX a, b & c	8 µg/cada uno

pALEX2-Ca

pALEX2-Ca is a new generation of CASCADETM expression vector for the production of C-terminal fusion to C-LYTAG sequence.

The C-LYTAG allows high degree protein purification using cost convenient chromatography supports (C-LYTRAP is more than five times less expensive than other commercial resins). This tag also provides improved solubility to recombinant proteins compared to His.tag and it is compatible with the purification of metalloproteins.

The main advantages of the expression and purification system are:

• - Low basal expression level facilitating cloning of toxic proteins and the stability of recombinant strains.
• - The CASCADETM system function does not require a particular media formulation.
• - Active at low growth temperature.
• - Higher level of purification than His-tag.
• - Low cost of main consumables (inducer, support, etc) allows cost efficient scaling up the production.
• - Compatibility with mini Tn5 delivery vectors for the construction of stable strains.

pALEX2-Ca vector derives from the new vector pALEX1a, therefore includes multiple cloning site of this plasmid with single targets for a wide variety of different restriction enzymes (for more details, consult the vector map).

NOTE: C-LYTAG Purification System is shipped at room temperature. Upon arrival, store the components according to the instructions in the table above.

* CASCADE^TM is a trademark of Active Motif, Inc., Carlsbad. The CASCADE™ expression system is patented and licensed by Active Motif, Inc. Commercial license available. Please contact us if you want more information about the license agreement.

pALEX1a, b and c vectors

pALEX1a, b and c are highly improved CASCADETM 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 (See cloning section).

REF.	PRODUCT	FORMAT
EV-3439	pALEX1 a	8 µg
EV-3440	pALEX1 b	8 µg
EV-3441	pALEX1 c	8 µg
EV-3462	pALEX1 a, b & c	8 µg/cada uno

pALEX2a, b and c vectors

The new pALEX2 vector series is designed to be used with the CASCADETM system for the expression, purification and immobilization of proteins fused in their N-termini to a shortened and improved version of C-LYTAG, 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 (See cloning section).

REF.	PRODUCT	FORMAT
EV-4643	pALEX2 a	8 µg
EV-4644	pALEX2 b	8 µg
EV-4645	pALEX2 c	8 µg
EV-4658	pALEX2 a, b & c	8 µg/cada uno