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  • pMYB5 Control Plasmid

    Discontinued Date

    11/01/2012
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    Categories:
    Discontinued Products

    Description

    pMYB5 is a control plasmid for the IMPACT™ Kit (NEB #E6901) (1,2). This plasmid carries the E. coli malE gene, encoding the maltose binding protein (MBP)(3), fused in-frame to the coding region of the Sce VMA intein-chitin binding domain (55 kDa)(1,4). pMYB5 can be used to test plasmid transformation, cell culture, induction and purification procedures. After induction with 0.3 mM IPTG at 30°C for 3 hours (or 15°C for 12-16 hours), 100 ml of cells should yield 2-3 mg of a 97 kDa fusion protein. After chitin column purification and cleavage, approximately 1.0-1.5 mg of the MBP (42 kDa) is usually obtained. This double stranded vector is 8602 bp in length.

    Advantages and Features

    Features

    • Expression of the fusion gene is under the control of an IPTG -inducible T7 promoter (5).
    • E. coli strains T7 Express Competent E. coli (High Efficiency) (NEB #C2566) or BL21(DE3) Competent E. coli (NEB #C2527) and derivatives can be used as expression hosts.
    • A pTYB1 derivative.
    • Ampicillin resistance.

    Properties and Usage

    Affinity Tag

    Other

    Storage Temperature

    -20°C

    Sequence Files

    Fasta  GenBank  

    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. Chong, S., Shao, Y., Paulus, H. Benner, J., Perler F.B. and Xu, M.-Q. (1996). Protein splicing involving the Saccharomyces, cerevisiae 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.
    3. Guan, C., Li, P., Riggs, P.D. and Inouye, H. (1988). Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene. 67, 21-30.
    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, 45-59.

    Interactive Tools

    Application Notes

    Material Safety Data Sheets

    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.