IMPACT™

Protein Expression

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.

References

  1. Chong, S. et al. (1997) Gene, 192, 277–281. PMID: 9224900  
  2. Chong, S. et al. (1998) Nucl. Acids Res. 26, 5109–5115. PMID: 9801307 
  3. Evans, T.C. et al. (1998) Protein Science, 7, 2256–2264. PMID: 9827992
  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
  9. Sun, L. et al. (2004) BioTechniques, 37, 430–443.
  10. Sun, L. et al. (2007) Methods, 42, 220–226. PMID: 17532508

IMPACT™ is a trademark of New England Biolabs, Inc.

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Advantages

  • 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)

Expression and Purification of E. coli Maltose-Binding Protein (MBP) Using the pTXB1 Vector (pMXB10)


Lane 1: uninduced cell extract.
Lane 2: induced cell extract showing expressed fusion protein.
Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C.
Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703)