Product Class: Kit

IMPACT™ Kit

Product Introduction

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
Catalog # Size Concentration
E6901S 1.0 set

Product Information

Description

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.
This product is related to the following categories:
IMPACT System,
Bacterial E. coli Protein Expression Products,
Chitin Purification (CBD-tag),
Affinity Purification Products,
Protein Purification Products,
Protein Expression Products,
Protein Labeling Products,
This product can be used in the following applications:
Affinity Purification & Expression Tags,
Protein Purification,
IMPACT Affinity Tag,
Peptide Ligation,
Protein Expression in E. Coli,
Protein Expression

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

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

References

  1. Chong, S. et al. (1997). Gene. 192, 277-281.
  2. Chong, S. et al. (1998). Nucl. Acids Res.. 26, 5109-5115.
  3. Evans, T.C. et al. (1998). Protein Sci.. 7, 2256-2264.
  4. Watanabe, T. et al. (1994). J. Bacteriol.. 176, 4465-4472.
  5. Muir, T.W. et al. (1998). Proc. Natl. Acad. Sci. USA. 95, 6705-6710.

Protocols, Manuals & Usage

Protocols

  1. Construction of the Fusion Plasmid (E6901)
  2. Primer Design for Restriction Enzyme Cloning (E6901)
  3. Fusion Constructs (E6901)
  4. Fusion Protein Expression (E6901)
  5. Affinity Purification and On-column Cleavage (E6901)
  6. Simplified Expression and Purification Protocol (E6901)
  7. Preparation of Media and Solutions (E6901)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What systems does NEB offer for protein expression and purification?
  2. What is IMPACT?
  3. What are the advantages of the IMPACT System?
  4. What vectors are included in the IMPACT kit?
  5. If my target protein is sensitive to DTT , which vector(s) should I use?
  6. What is Intein-mediated Protein Ligation (IPL)?
  7. What has IPL been used for?
  8. Where can find all IMPACT FAQs?
  9. What gravity column do you recommend?
  10. What sequencing primers do you recommend for pTXB1 and pTYB21 vectors?

Quality, Safety & Legal

Quality Assurance Statement

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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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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