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  • pTYB22 Vector

    Discontinued Date

    11/01/2012
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    Discontinued Products

    Description

    pTYB22 is an E. coli cloning and expression vector (7511 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). It is an 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 a C-terminal fusion vector 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 pTXB1 (NEB #N6707), pTXB3 (NEB #N6708), pTYB1 (NEB #N6701), pTYB2 (NEB #N6702), pTYB3 (NEB #N6703) or pTYB4 (NEB #N6704).

    Advantages and Features

    Features

    • The multiple cloning site (MCS) is compatible with the multiple cloning sites of vectors in the pMAL Protein Fusion and Purification System (NEB #E8200) and the K. lactis Protein Expression Kit (NEB #E1000).
    • 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 pTYB22 adds extra three residues (Ala-Gly-His) to the N-terminus of the target protein. 
    • A stop codon should be included in the reverse primer.
    • Compatible sites are present in both pTYB22 (eg. NdeI and EcoRV) and pTYB2 (eg. NdeI and SmaI; 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.
    • A pBR322 derivative with a ColE1 replication origin.
    • Expression of the fusion gene is under the control of the T7/lac 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.,T7 Express Competent E. coli (High Efficiency),  (NEB #C2566)  or BL21(DE3) Competent E. coli (NEB #C2527) and derivatives].
    • Ampicillin resistance.
    • When pTYB21 or pTYB22 is used, a small peptide (15 amino acids, 1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. It cannot be detected on a regular SDS-PAGE and can be dialyzed out.
    • 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 #N0315) is available.

    Properties and Usage

    Affinity Tag

    Chitin-Binding Domain (CBD)

    Storage Temperature

    -20°C

    References

    1. Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., 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.
    2. 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.
    3. 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, 10567-77.
    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. 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, 45-49.

    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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