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.
- Affinity Purification and On-column Cleavage (E6901)
- Simplified Expression and Purification Protocol (E6901)
- High Efficiency Transformation Protocol (C3013)
- High Efficiency Transformation Protocol (C3016)
- Protein Expression Using BL21(DE3) (C2527)
- Protocol for Expression Using T7 Express (C2566)
- Protocol for Expression Using T7 Express lysY (C3010)
- Protocol for Expression Using T7 Express Iq (C3016)
- Protein Expression with T7 Express strains
- High Efficiency Transformation Protocol (C2529)
- Preparation of Media and Solutions (E6901)
- Transformation Protocol (C2528)
- Expression Using SHuffle®
- 5 Minute Transformation Protocol (C2527)
- Protein Expression Using Lemo21(DE3) (C2528)
- 5 Minute Transformation Protocol (C2528)
- 5 Minute Transformation Protocol (C3016)
- Protocol for Removal of IMAC Contaminating Proteins (C2529)
- Protein Expression Using NiCo21(DE3) (C2529)
- Fusion Constructs (E6901)
- Transformation Protocol for BL21(DE3) Competent Cells (C2527)
- Construction of the Fusion Plasmid (E6901)
- Protocol for Expression Using T7 Express lysY/Iq (C3013)
- 5 Minute Transformation Protocol (C2529)
- 5 Minute Transformation Protocol (C3013)
- 5 Minute Transformation Protocol (C2566)
- High Efficiency Transformation Protocol (C2566)
- 5 Minute Transformation Protocol (C3010)
- Fusion Protein Expression (E6901)
- High Efficiency Transformation Protocol
- Primer Design for Restriction Enzyme Cloning (E6901)
Avoid Common Obstacles in Protein Expression
Read how to avoid common obstacles in protein expression that prevent interactions with cellular machinery.
- Competent Cell Brochure
- Protein Expression & Purification Brochure
- Purification Beads, Columns and Resins Brochure
- Competent Cell Product Comparison
- IMPACT™ Vectors and Applications
- Protein Expression and Purification Selection Chart
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.