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  • IMPACT™ Kit

    Description

    The IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag (1). It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step, a native recombinant protein without the use of a protease. Each intein tag contains a chitin binding domain (CBD) for the affinity purification of the fusion protein on a chitin resin (2-4). Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the target protein from the intein tag (Figures 1,2). The vectors included in this kit allow for the fusion of the target protein at its C-terminus (pTXB1) (3,5) or at its N-terminus (pTYB21) (4,6) to the intein tag.

    In addition, with the use of pTXB1, native recombinant proteins that possess a reactive C-terminal thioester can be isolated for applications, including protein semisynthesis and site-specific labeling [3,7, Intein Mediated Protein Ligation (IPL, Appendix I)].



    Figure 1:
    Purification of Maltose Binding Protein (MBP) in a single affinity purification step: Lane 1: uninduced cell extract. Lane 2: induced cell extract showing expressed fusion protein. Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C. Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703).
    Figure 2:
    Schematic of the IMPACT System.
    Figure 3:
    Intein-mediated Protein Ligation (IPL).
    Figure 4:
    Polylinkers in the vectors pTXB1 and pTYB21.
    ▼ indicates intein cleavage site.
    Figure 5:
    Flow chart for Protein Expression and Purification using the IMPACT System. Sample collection for analysis by SDS-PAGE is indicated.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pTXB1 Vector-20200 μg/ml
    pTYB21 Vector-20200 μg/ml
    pMXB10 Control Plasmid-200.2 mg/ml
    Chitin Resin4
    1M DTT1,000 mM
    E. coli B ER2566
    Blue Loading Buffer-203X
    Anti-CBD Monoclonal Antibody-202 mg/ml

    Advantages and Features

    Features

    • Single-column purification without the use of proteases to remove the affinity tag
    • Able to produce target protein without vector–derived amino acids
    • Fusion to either C-terminus or N-terminus of target protein
    • Isolation of proteins with or without an N-terminal methionine residue
    • Ligation and labeling of recombinant proteins
    • T7 promoter-driven system to achieve high levels of expression and tight transcriptional control in E. coli

    Properties and Usage

    Storage Temperature

    -20°C

    References

    1. Chong, S. et al. (1997). Gene. 192, 277-281.
    2. Chong, S. et al. (1998). Nucl. Acids Res.. 26, 5109-5115.
    3. Evans, T.C. et al. (1998). Protein Sci.. 7, 2256-2264.
    4. Watanabe, T. et al. (1994). J. Bacteriol.. 176, 4465-4472.
    5. Muir, T.W. et al. (1998). Proc. Natl. Acad. Sci. USA. 95, 6705-6710.

    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.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].
    1. What systems does NEB offer for protein expression and purification?
    2. What is IMPACT?
    3. What are the advantages of the IMPACT System?
    4. What vectors are included in the IMPACT kit?
    5. If my target protein is sensitive to DTT , which vector(s) should I use?
    6. What is Intein-mediated Protein Ligation (IPL)?
    7. What has IPL been used for?
    8. IMPACT FAQs
    1. Construction of the Fusion Plasmid (E6901)
    2. Primer Design for Restriction Enzyme Cloning (E6901)
    3. Cloning Using Gibson Assembly (E6901)
    4. Fusion Constructs (E6901)
    5. Fusion Protein Expression (E6901)
    6. Affinity Purification and On-column Cleavage (E6901)
    7. Simplified Expression and Purification Protocol (E6901)
    8. Preparation of Media and Solutions (E6901)

    Selection Tools

    Application Notes