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  • pMXB10 Control Plasmid

    Discontinued Date

    11/01/2012
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    Discontinued Products

    Description

    pMXB10 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, 42 kDa) (3), fused in-frame to the coding region of the Mxe gyrase A intein-chitin binding domain (28 kDa) from pTXB1 (2,4,5). pMXB10 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 70 kDa fusion protein. After chitin column purification and cleavage, approximately 1.5-2.0 mg of MBP is usually obtained. This double stranded vector is 7813 bp in length.

    Highlights

    • Expression of the fusion gene is under the control of an IPTG -inducible T7 promoter (5).
    • 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 pTXB1 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. Evans, T.C., Benner, J., and Xu, M.-Q. (1998). Semisynthesis of cytotoxic proteins using a modified protein splicing element. Prot. Sci.. 7(11), 2256-2264.
    3. Guan, C. D. 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. Southworth, M.W., Amaya, K., Evans, J., T.C., Xu, M.-Q. and Perler, F.B. (1999). Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein.. BioTechniques. 27, 110-120.
    5. 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.
    6. 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.
    1. What vectors are included in the IMPACT kit?

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