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IMPACT™ Kit

Catalog # Concentration Size List Price Quantity Your Price
E6901S 1 set $718.00
$646.20
Catalog # Size List Price Your Price
E6901S 1 set $718.00
$646.20
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

The IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements, termed inteins, to separate the target protein from the affinity tag

  • 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
  • Able to produce a target protein without vector–derived amino acids
  • Fusion to either C-terminus or N-terminus of target protein
  • Isolation of proteins with or without an N-terminal methionine residue
  • Ligation and labeling of recombinant proteins
  • T7 promoter-driven system to achieve high levels of expression and tight transcriptional control in E. coli
IMPACT Citations

The IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification strategy that utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag (1). 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. Each intein tag contains a chitin binding domain (CBD) for the affinity purification of the fusion protein on chitin resin (2–4). Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the target protein from the intein tag (Figures 1,2). The vectors included in this kit allow for the fusion of the target protein at its C-terminus (pTXB1) (3,5) or at its N-terminus (pTYB21) (4,6) to the intein tag.

In addition, with the use of pTXB1, native recombinant proteins that possess a reactive C-terminal thioester can be isolated for applications, including protein semisynthesis and site-specific labeling [3,7, Intein Mediated Protein Ligation (IPL, Appendix I)].



Figure 1: Purification of Maltose Binding Protein (MBP) in a single affinity purification step
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).
Figure 2: Schematic of the IMPACT System


Figure 3: Intein-mediated Protein Ligation (IPL)


Figure 4: Polylinkers in the vectors pTXB1 and pTYB21 
▼ indicates intein cleavage site.
Figure 5: Flow chart for Protein Expression and Purification using the IMPACT System
Sample collection for analysis by SDS-PAGE is indicated.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  IMPACT™ Kit E6901-1 -20 1 x 1 set
  Chitin Resin S6651S 4 1 x 20 ml
Features
  • Single-column purification without the use of proteases to remove the affinity tag
  • Able to produce target protein without vector–derived amino acids
  • Fusion to either C-terminus or N-terminus of target protein
  • Isolation of proteins with or without an N-terminal methionine residue
  • Ligation and labeling of recombinant proteins
  • T7 promoter-driven system to achieve high levels of expression and tight transcriptional control in E. coli

Properties & Usage



Companion Products
Materials Sold Separately
References
  • Chong, S. et al. (1997). Gene. 192, 277-281.
  • Chong, S. et al. (1998). Nucl. Acids Res.. 26, 5109-5115.
  • Evans, T.C. et al. (1998). Protein Sci.. 7, 2256-2264.
  • Watanabe, T. et al. (1994). J. Bacteriol.. 176, 4465-4472.
  • Muir, T.W. et al. (1998). Proc. Natl. Acad. Sci. USA. 95, 6705-6710.
Citations
Publications
  • Mauris, J.and Evans, T.C., Jr. (2010). A human PMS2 homologue from Aquifex aeolicus stimulates an ATP-dependent DNA helicase. J Biol Chem. 285(15), 11087-11092.PubMedID: 20129926
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
Legal And Disclaimer

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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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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