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IMPACT™ Kit |
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- 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
Description:
The IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) Kit utilizes engineered protein splicing elements (termed inteins) to purify recombinant proteins by a single column (1-3) (Figure 1). This kit distinguishes itself from other protein fusion systems by its ability to separate a recombinant protein from the affinity tag without the use of a protease.
The IMPACT Kit allows fusion of a tag consisting of the intein and the chitin binding domain (CBD) (4), to either the C-terminus (pTXB1) or the N-terminus (pTYB11) of the target protein (Figure 2). In the presence of thiols, such as DTT, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag. The pTXB1 vector can also be used to express and purify a protein with a C-terminal thioester for use in Intein-mediated Protein Ligation (IPL). The IPL reaction, also referred to as expressed protein ligation, allows for the ligation of a peptide or a protein with a N-terminal cysteine to a bacterially expressed protein with a C-terminal thioester through a native peptide bond (Figure 3) for use in protein labelling and semisynthesis (3,5).
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. Marker M is the Protein Marker, Broad Range (NEB #P7702).
Figure 2: Schematic of the IMPACT System.
Figure 3: Intein-mediated Protein Ligation (IPL).
Figure 4: IMPACT vectors and sequencing primers. All IMPACT Vectors contain the IPTG-inducible T7 promoter. Click here for detailed restriction maps.
CBD = chitin binding domain, MCS = multiple cloning site, ∇ = intein cleavage site(s).
Kit Components:
E. coli Strain ER2566
1, 4-Dithiothreitol (DTT)
Anti-Chitin Binding Domain Serum
Blue Loading Buffer
Chitin Beads
pMXB10 Control Plasmid
pTXB1 Vector
pTYB11 Vector
Storage Conditions
Storage Temperature:
-20°C
FAQs
- What is IMPACT?
- What are the advantages of the IMPACT System?
- What vectors are included in the IMPACT kit?
- If my target protein is sensitive to DTT , which vector(s) should I use?
- What is Intein-mediated Protein Ligation (IPL)?
- What has IPL been used for?
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.
Reagents Sold Separately
Anti-Chitin Binding Domain Serum
Chitin Beads
pMXB10 Control Plasmid
pTXB1 Vector
pTYB11 Vector
Companion Products
Anti-CBD Monoclonal Antibody
Bio-P1 (Lyophilized)
Chitin Magnetic Beads
Flu-P1 (Lyophilized)
Legal
Licenses/Patents/Disclaimers:
The buyer and user have a non-exclusive sublicense to use this system or any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.
Transfer of the host cells that contain the cloned copy of the T7 gene 1 to third parties is explic-itly prohibited. This limitation applies to E. coli strain ER2566 which is provided in combination with the IMPACT™ system or in combination with appropriate vectors for said system.
A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.
Commercial Laboratory Buyer and User: Use of the host cells that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase for any purpose other than in combination with the IMPACT™ system is explicitly prohibited.
Use of the host cells that contain the copy of the T7 gene 1, the gene for T7 RNA polymerase with any other vector(s) containing a T7 promoter to direct the production of RNA or protein requires a license from Brookhaven National Laboratory. Information about research-use or commercial-use license agreements may be obtained from the Office of Technology Transfer, Brookhaven National Laboratory, Building 475D, P.O. Box 5000, Upton, New York, 11973-5000; telephone: 631-344-7134, fax: 631-344-3729.
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