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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(4) Muir, T.W., Sondhi, D. and Cole, P.A. (1998) Proc. Natl. Acad. Sci. USA, 95, 6705–6710. PMID: 9618476
(5) Dubendorff, J.W. and Studier, F.W. (1991) J. Mol. Biol., 219, 45–59. PMID: 1902522
(6) Ghosh, I. et al. (2004) J. of Imm. Methods, 293, 85–95. PMID: 15541279
(7) Xu, J. et al. (2004) BioTechniques, 36, 976–978. PMID: 15211748
(8) Kochinyan, S. et al. (2007) BioTechniques, 42, 63–69. PMID: 17269486
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(10) Sun, L. et al. (2007) Methods, 42, 220–226. PMID: 17532508
IMPACT™ is a trademark of New England Biolabs, Inc.
FAQs for IMPACT™
Protocols for IMPACT™
- Fusion Constructs (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Preparation of Media and Solutions (E6901)
- Simplified Expression and Purification Protocol (E6901)
- Construction of the Fusion Plasmid (E6901)
- Affinity Purification and On-column Cleavage (E6901)
- Isolation of CBD-fusion protein using Chitin Magnetic Beads
- Fusion Protein Expression (E6901)
- Isolation of MBP-fusion protein using Amylose Magnetic Beads
- Protein Expression with T7 Strains
- Expression Using SHuffle®
Application Notes for IMPACT™
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
- 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)