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  • pMYB5 Control Plasmid

    Discontinued Date

    11/01/2012
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    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

    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.

    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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    Application Notes