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  • pTYB2 Vector

    Discontinued Date

    11/01/2012
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    Discontinued Products

    Description

    pTYB2 is an E. coli cloning and expression vector (7474 bp) used in the IMPACT™ Kit (NEB #E6901) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into the 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.

    For fusion of the N-terminus of the target protein to the intein tag, use pTYB21 (NEB #N6709) or pTYB22 (NEB #N6710). This vector can be used in conjuction with pTYB22 (NEB #N6710) to test which fusion constructin (N-terminal or C-terminal) maximizes the expression and yield of a target protein (3).

    Advantages and Features

    Features

    • The NdeI site in the polylinker contains an ATG sequence for translation initiation.
    • The SmaI site is used for cloning the 3´ end of the target gene and will yield a target protein with a single glycine residue added to its C-terminus after cleavage of the intein.
    • A pBR322 derivative with a ColE1 replication origin.
    • Expression of the fusion gene is under the control of an IPTG-inducible T7 promoter (4).
    • Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., T7 Express Competent E. coli (High Efficiency) NEB #C2566), BL21(DE3) Competent E. coli (NEB #C2527) and derivatives].
    • Ampicillin resistance.
    • M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid. M13K07 helper phage is available from (NEB #N0315).
    • 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.
    • The sites in the polylinker region are identical to or compatible with (i.e., NheI of pTYB2 and SpeI of pTYB12) those of pTYB12 (NEB #N6902). This allows the same amplified target gene 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.
    • Companion vectors (pTYB1, pTYB3, pTYB4) differ only in the sites present in the polylinker.

    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. 192, 277-281.
    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., 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.
    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.

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    Application Notes

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.