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  • pTYB3 Vector

    Discontinued Date

    11/01/2012
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    Discontinued Products

    Description

    pTYB3 is an E. coli expression vector (7,477 bp) used in the IMPACT™ Kit (NEB #E6901) (1). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into a polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa) (1,2). This results in the fusion of the C-terminus of the target protein to the N-terminus of the intein tag. Thiol-induced self-cleavage of the intein releases the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein (3).

    For fusion of the N-terminus of the target protein to the intein tag, use pTYB21 (NEB #N6709) or pTYB22 (NEB #N6710).

    Advantages and Features

    Features

    • The NcoI site in the polylinker contains an ATG sequence for translation initiation.
    • 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.
    • Unique sites are indicated in bold.
    • Expression of the fusion gene is under the control of an IPTG-inducible T7 promoter (4).
    • 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).
    • A pBR322 derivative with a ColE1 replication origin.
    • 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 vectors (pTYB1, pTYB2, pTYB4) differ only in the sites present in the polylinker.
    • 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

    Chitin-Binding Domain (CBD)

    Storage Temperature

    -20°C

    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. 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.
    3. Chong, S., Shao, Y., Paulus, H., Benner, J., Perler F.B. and Xu, M.-Q. (1996). Protein splicing involving the Saccharomyces, <em>cerevisiae</em> VMA intein: the steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J. Biol. Chem. 271, 22159-22168.
    4. 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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

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