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Prices are in US dollars and valid only for US orders.
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Description:
pTXB3 is an E. coli expression vector in the IMPACT™ Protein Purification System (1,2). It is designed for the in-frame insertion of a target gene into the polylinker upstream of the Mxe intein/chitin binding domain (27 kDa) (2,3,4,5). The fusion protein is bound to chitin beads and the thiol-induced cleavage activity of the intein releases the target protein. pTXB vectors are recommended for use in intein-mediated protein ligation and C-terminal labeling (2).This double-stranded vector is 6,706 base pairs in length.
Source:
pTXB3 contains the intein (198 amino acids) from the Mycobacterium xenopi GyrA gene (2,3,4,5).
Advantages:- The NcoI site in the polylinker contains an ATG sequence for translation initiation. Unique sites are indicated in bold. SalI site is not unique.
- The SapI site should be used for cloning of the 3´ end of the insert. Use of the SapI site allows cloning of the target protein adjacent to the intein, resulting in cleavage of the target protein without any additional amino acids at its C-terminus.
- Expression of the fusion gene is under the control of an IPTG-inducible T7 promoter (6).
- A pBR322 derivative with a ColE1 reprication origin.
- Origin of DNA replication from bacteriophage M13, which allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid (M13K07 Helper phage, NEB #N0315).
- Ampicillin resistance.
- T7 Universal Primer (NEB #S1248) and Mxe Intein Reverse II Primer (NEB #S1285) are available for sequencing the insert.
- Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein. The cleavage reaction may be induced by thiol reagent or temperature/pH shift.
- Companion vector pTXB1 (NEB #N6707) contains an NdeI site in place of NcoI.
- A wide range of E. coli host strains: T7 Express Cells or BL21 (DE3) and derivatives.
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:- E. coli strain ER2566 is only available to purchasers of the IMPACT™ System or replacement vectors.
References
- 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, 271-281.
- Evans, T.C., Benner, J. and Xu, M.-Q. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci., 7, 2256-2264.
- Watanabe, T., Ito, Y., Yamada, T., Hasmimoto, 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.
- Telenti, A., Southworth, M., Alcaide, F., Daugelat, S., Jacobs, W.R. Jr. and Perler, F.B. (1997) The Mycobacterium xenopi GyrA protein splicing element: Characterization of a minimal intein. J. Bacteriol., 179, 6378-6382.
- 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.
- Southworth, M.W., Amaya, K., Evans, J.,T.C., Xu, M.-Q. and Perler, F.B. (1999) Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein.. Biotechniques, 27, 110-120.
Companion Products
pTXB1 Vector
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,496,714
New England Biolabs, Inc.: U.S. Patent No. 5,834,247
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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