IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) is a novel protein purification system which utilizes the inducible self-cleavage activity of a protein splicing element (termed intein) to separate the target protein from the affinity tag. 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 (1,2). A target protein is fused to a tag consisting of the intein and the chitin binding domain which allows affinity purification of the fusion protein on a chitin column. In the presence of thiols such as DTT or 2-mercaptoethanesulfonic acid (MESNA), the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag resulting in a single-column purification of the target protein. In addition, with the use of pTXB1, native recombinant proteins that possess a reactive C-terminal thioester can be isolated for applications including protein ligation, semisynthesis and site-specific labeling (3,4).
The IMPACT™ Kit (NEB #E6901) contains expression vectors which allow fusion of the cleavable intein tag to either the C-terminus or N-terminus of the target protein. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein.
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IMPACT™ is a trademark of New England Biolabs, Inc.
- Affinity Purification and On-column Cleavage (E6901)
- Construction of the Fusion Plasmid (E6901)
- Fusion Protein Expression (E6901)
- Isolation of CBD-fusion protein using Chitin Magnetic Beads
- Isolation of MBP-fusion protein using Amylose Magnetic Beads
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Simplified Expression and Purification Protocol (E6901)
- Protein Expression with T7 Express strains
- Expression Using SHuffle®
- Fusion Constructs (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
- Competent Cell Product Comparison
- IMPACT™ Vectors and Applications
- Protein Expression and Purification Selection Chart
- Yields proteins with native sequence
- One-step affinity purification with no additional steps to remove affinity tag
- Release from the fusion partner without the use of proteases
- Fusion to either the C-terminus or N-terminus of the target protein
- The ability to label the C-terminus of the target protein
- T7 Promoter for higher levels of expression and tight transcriptional control
- The ability to introduce non-coded amino acids, fatty acids or chemically modified amino acid residues (segmental labeling of proteins for NMR analysis)
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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.