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
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