Protein Expression
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  • IMPACT™ Protein Expression

    Removal of an affinity tag from a purified recombinant protein is commonly performed using digestion with site-specific proteases. A drawback to this approach is that the released target protein must be purified from the liberated tag and the protease using additional chromatography steps. Additionally, proteases can occasionally cleave off-site which can be detrimental to the target protein. Inteins are protein segments that auto-catalyze their excision from proteins, offering an alternative to protease cleavage. Used in conjunction with a strong-binding affinity tag, an intein can permit one-step purification of a target protein without the dangers of off-site protease cleavage. Additionally, an intein can be used in a manner that leaves no non-native amino acids on the target protein following its excision.

    The IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) Kit (NEB #E6901) is an E.coli protein expression system that permits expression of target proteins carrying an intein-chitin binding domain (intein-CBD) tag for one-step purification using a chitin resin. Upon passage of a cell lysate over chitin resin, fusion proteins become immobilized. The target protein can be specifically released from the fusion protein through induction of intein cleavage by the addition of a thiol-containing buffer (e.g. DTT) or a pH shift (from 8.5 to 6). The target protein then elutes from the column while the CBD-intein portion remains bound. Different IMPACT vectors permit the intein-CBD tag to be added to the N- or C-terminus of a target protein.

    Another unique attribute of the IMPACT system is that it can produce proteins that can be labeled or ligated in vitro using Intein-mediated Protein Ligation (IPL). A target protein expressed with a C-terminal intein-CBD tag retains a thioester group on its C-terminus after intein cleavage. IPL permits a new peptide bond to form between the expressed protein’s C-terminal thioester and any heterologous peptide or protein having an N-terminal cysteine.

    IMPACT™ is a trademark of New England Biolabs, Inc.

    Schematic Illustration of IMPACT System

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    Protocols for IMPACT™ Protein Expression

    Expression and Purification of E. coli Maltose Binding Protein MBP Using the IMPACT™ System

    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)