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.
FAQs for IMPACT™ Protein Expression
Protocols for IMPACT™ Protein Expression
- 5 Minute Transformation Protocol (C2527)
- 5 Minute Transformation Protocol (C2528)
- 5 Minute Transformation Protocol (C2529)
- 5 Minute Transformation Protocol (C2566)
- 5 Minute Transformation Protocol (C3010)
- 5 Minute Transformation Protocol (C3013)
- 5 Minute Transformation Protocol (C3016)
- Affinity Purification and On-column Cleavage (E6901)
- Construction of the Fusion Plasmid (E6901)
- Expression Using SHuffle®
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C2529)
- High Efficiency Transformation Protocol (C2566)
- High Efficiency Transformation Protocol (C3013)
- High Efficiency Transformation Protocol (C3016)
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protein Expression Using BL21(DE3) (C2527)
- Protein Expression Using Lemo21(DE3) (C2528)
- Protein Expression Using NiCo21(DE3) (C2529)
- Protein Expression with T7 Strains
- Protocol for Expression Using T7 Express (C2566)
- Protocol for Expression Using T7 Express Iq (C3016)
- Protocol for Expression Using T7 Express lysY (C3010)
- Protocol for Expression Using T7 Express lysY/Iq (C3013)
- Protocol for Removal of IMAC Contaminating Proteins (C2529)
- Simplified Expression and Purification Protocol (E6901)
- Transformation Protocol (C2528)
- Transformation Protocol for BL21(DE3) Competent Cells (C2527)
Application Notes IMPACT™ Protein Expression
Bypassing Common Obstacles in Protein Expression
Competent Cells Brochure
The Competent Cells brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
- Competent Cell Product Comparison
- IMPACT™ Vectors and Applications
- Protein Expression and Purification Selection Chart
- Convenient Formats of Competent Cells
Other Tools & Resources
Expression and Purification of E. coli Maltose Binding Protein MBP Using the IMPACT™ System