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pTYB12 Vector |
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Prices are in US dollars and valid only for US orders.
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Description:
pTYB12 is an E. coli cloning and expression vector (7417 bp) used in the IMPACT™ Protein Purification System which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). It is a N-terminal fusion vector designed for in-frame insertion of a target gene into the polylinker, downstream of the intein tag (the Sce VMA intein/chitin domain, 55 kDa)(3,4). This allows the N-terminus of the target protein to be fused to the intein tag. The self-cleavage activity of the intein allows the release of the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein. This vector can be used in conjuction with pTYB2 (NEB #N6702S) to test which fusion construction (N-terminal or C-terminal) maximizes the expression and yield of a target protein.
For the fusion of the C-terminus of the target protein to the intein tag, use pTYB1 (NEB #N6701S), pTYB2 (NEB #N6702S), pTYB3 (NEB #N6703S), or pTYB4 (NEB #N6704S).
Source:
pTYB12 is isolated from an E. coli strain (r-m-) by a standard plasmid purification procedure.
Advantages:- The sites in the polylinker region are identical to or compatible with (i.e. SpeI of pTYB12 and NheI of pTYB2) those of pTYB2 (NEB #N6702S). This allows the same amplified target gene fragment to be cloned into either vector for optimizing protein expression.
- Vector derived residues may be present at the N and/or C-termini of the target proteins.
- Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein. The cleavage reaction can be induced by thiol reagent or temperature/pH shift.
- After the cleavage of the intein tag, a target protein is obtained with extra residue(s) added to N-terminus. For instance, cloning the 5´ end of a target gene using NdeI site in pTYB12 adds extra three residues (Ala-Gly-His) to the N-terminus of the target protein.
- A pBR322 derivative with a ColE1 replication origin.
- Expression of the fusion gene is under the control of the T7 promoter and can be induced by IPTG due to the presence of a lacI gene (5).
- Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., ER2566 or BL21(DE3) and derivatives].
- Ampicillin resistence.
- Origin of DNA replication from the bacteriophage M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid. M13K07 Helper Phage (NEB #N0315S) is available.
- Intein Forward Primer (NEB #S1263S) and T7 Terminator Reverse Primer (NEB #S1271S) are available for sequencing the target gene.
Concentration:
200 μg/ml
Storage Conditions
Storage Conditions:
10 mM Tris-HCl
1 mM EDTA
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Product ER2566 is only available to purchasers of the IMPACT™ System or replacement vectors.
References
- Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q. and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res., 26, 5109-5115.
- Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H. and Xu, M.-Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene, 192, 277-281.
- Chong, S., Williams, K.S., Wotkowicz, C. and Xu, M.-Q. (1998) Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem., 273, 10567-10577.
- Watanabe, T., Ito, Y., Yamada, T., Hashimoto, M., Sekine, S. and Tanaka, H. (1994) The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol., 176, 4465-4472.
- Dubendorff, J.W. and Studier, F.W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol., 219, 45-59.
Companion Products
pKYB1 Vector
pTYB1 Vector
pTYB11 Vector
pTYB2 Vector
pTYB3 Vector
pTYB4 Vector
Legal
Research Use Assurance:
The buyer and user have a nonexclusive sublicense to use this system for any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.
Transfer of this vector in host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited.
Licenses/Patents/Disclaimers:
Notice to Buyer/User: the buyer/user has a non-exclusive license to use this system or any components thereof for RESEARCH PURPOSES ONLY. See Research Use Assurance Statement for details on terms of the license granted hereunder.
A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.:
Commercial Laboratory Buyer/User: Use of the vector in host cells that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase for any purpose other than in combination with the IMPACT™ System is explicitly prohibited.
Use of the host cells that contain the copy of the T7 gene 1, the gene for T7 RNA polymerase with any other vector(s) containing a T7 promoter to direct the production of RNA or protein requires a license from Brookhaven National Laboratory. Information about research-use or commercial-use license agreements may be obtained from the Office of Technology Transfer, Brookhaven National Laboratory, Building 475D, P.O. Box 5000, Upton, New York 11973-5000; Tel. 631-344-7134, Fax: 631-344-3729.
You may refuse this non-exclusive research license agreement by returning the enclosed materials unused. By keeping or using the enclosed materials, you agree to be bound by the terms of this sub-license.
Note: E. coli strain ER2566 is only available to purchasers of the IMPACT™ System or replacement vectors.
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