pTXB1 Vector

Description

pTXB1 is an E. coli expression vector in the IMPACT™ Kit (NEB #E6901) (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.

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Advantages and Features

Features

• The NdeI 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 ColE1replication 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.
• 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 pTXB3 (NEB #N6708) contains an NcoI site in place of NdeI.
• A wide range of E. coli host strains: T7 Express Competent E. coli (High Efficiency) (NEB #C2566) or BL21(DE3) Competent E. coli (NEB #C2527) and derivatives.

Properties and Usage

Affinity Tag

Storage Temperature

Related Products

Companion Products

• IMPACT™ Kit
• pTYB21 Vector
• pMXB10 Control Plasmid
• pTWIN1 Vector

References

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.