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This product is currently
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This product has been discontinued and replaced by IMPACT Kit (E6901)
Description:
IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) utilizes the inducible self-cleavage activity of engineered protein splicing elements (termed inteins) to purify recombinant proteins by a single affinity column (1-4) (Figures 1 and 2). This system 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-CN Protein Fusion and Purification System contains four expression vectors (pTYB vectors) which allow fusion of a bifunctional tag, consisting of the intein and the chitin binding domain (5), to either the C-terminus (pTYB1,2) or N-terminus (pTYB11,12) of the target protein. In the presence of thiols such as DTT, β-mercaptoethanol or cysteine, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag.
Both pTYB1 and pTYB11 contain a SapI cloning site which allows the target gene to be cloned immediately adjacent to the cleavage site of the intein tag; this results in the purification of a target protein without any extra non-native residues attached to its terminus after cleavage. Use of pTYB2 or pTYB12 yields a target protein with extra residue(s) added to its C-terminus or N-terminus, respectively. Addition of extra residues may help the cleavage reaction for some target proteins. pTYB2 and pTYB12 contain the same or compatible restriction sites in the multiple cloning region. This allows cloning the same target gene fragment into both vectors. pTYB1 and pTYB2 use ATG of the NdeI site in the multiple cloning region for translation initiation.
Vectors sold separately: pTYB3 and pTYB4 contain an NcoI site in place of the NdeI site in pTYB1 and pTYB2, respectively. pKYB1 is identical to pTYB1 except that it carries kanamycin resistance instead of ampicillin resistance. pTXB1 and pTXB3 contain a mini-intein with alternative preferred residues at the cleavage site (10,11).
Figure 1: Schematic of the IMPACT-CN System.
Figure 2: Purification of biologically active T4 DNA Ligase with >98% purity in one step: Std: NEB Broad Range Protein Marker. Lane 1 (Clone): uninduced cell extract. Lane 2 (Express): induced cell extract showing expressed fusion protein. Lane 3 (Purify): T4 DNA Ligase fractions eluted after inducing cleavage overnight at 4°C.
Advantages:- Flexibility-allows fusion to either the C-terminus or N-terminus of the target protein
- Yields proteins with native sequence
- Release of fusion partner without the use of proteases
- One-step affinity purification-no additional steps to remove affinity tag
- T7 Promoter for higher levels of expression
- Tight transcriptional control
- The ability to label the C-terminus of the target protein
Kit Components:
E. coli Strain ER2566
1, 4-Dithiothreitol (DTT)
Anti-Chitin Binding Domain Serum
Blue Loading Buffer
Chitin Beads
Intein Forward Primer (24-mer)
Intein Reverse Primer (24-mer)
pMYB5 Control Plasmid
pTYB1 Vector
pTYB11 Vector
pTYB12 Vector
pTYB2 Vector
T7 Terminator Reverse Primer (19-mer)
T7 Universal Primer (20-mer)
Storage Conditions
Storage Temperature:
-20°C
Notes
General notes:- Cloning Vectors: The pTYB vectors are used for cloning and expression of recombinant proteins in E. coli (Figure 2). pTYB1 and pTYB2 are C-terminal fusion vectors in which the C-terminus of the target protein is fused to the intein tag. pTYB11 and pTYB12 are N-terminal fusion vectors in which the N-terminus of the target protein is fused to the intein tag; when pTYB11 and pTYB12 are used, a small peptide (1.2 kDa) is also cleaved from the intein tag and co-eluted with the target protein. It cannot be detected on a regular SDS-PAGE gel but can be separated from the target protein by dialysis. pTYB1 and pTYB2 use ATG of the NdeI site in the multiple cloning region for translation initiation. Both pTYB1 and pTYB11 contain a SapI cloning site which allows the target gene to be cloned adjacent to the cleavage site of the intein tag; this results in the purification of a target protein without any extra non-native residues attached to its terminus. pTYB2 and pTYB12 contain the same or compatible restriction sites in the multiple cloning region. This allows fusion of the intein tag to either termini of the same amplified target gene.
Use of pTYB2 or pTYB12 yields a target protein with extra residue(s) added to its C-terminus or N-terminus, repectively, after the cleavage of the intein tag. For instance, cloning the 3´ end of a target gene using the SmaI site in pTYB2 adds an extra glycine residue to the C-terminus of the target protein. Likewise, cloning the 5´ end of a target gene using the NdeI site in pTYB12 adds four extra residues (Ala-Gly-His-Met) to the N-terminus the target protein.
The pTYB vectors use a T7 promoter and the lac I gene to provide stringent control of the fusion gene expression. Binding of the lac repressor to the lac operator sequence immediately downstream of the T7 promoter suppresses basal expression of the fusion gene in the absence of IPTG induction. The four tandem copies of the E.coli transcription terminator (rrnB T1) placed upstream of the promoter minimize background transcription. The vectors also contain the origin of DNA replication from bacteriophage M13, which allows for the production of single-stranded DNA by helper phage (M13KO7 Helper Phage, NEB #N0315S) superinfection of cells bearing the plasmid. pTYB vectors carry the Ampr gene marker (the bla gene), which conveys ampicillin resistance to the host strain. - Sequencing Primers: Three primers (200 picomoles of each) are included for sequencing the target gene cloned in the multiple cloning region of the pTYB vectors. The T7 Universal Primer and Intein Reverse Primer are used for sequencing a target gene cloned in the C-terminal fusion vectors (pTYB1 and pTYB2). The intein forward primer is complementary to the intein sequence 117-141 nucleotides upstream from the intein cleavage site and used for sequencing a target gene cloned in the N-terminal fusion vectors (pTYB11 and pTYB12).
- Chitin Beads: An affinity matrix used for the isolation of the fusion precursor containing the target protein. 20 ml of chitin beads (~ 50-100 µm in size) are supplied as a 38 ml slurry in 20% ethanol. The binding capacity, which has been tested using the control vector pMYB5, is 2 mg of eluted MBP protein per ml of chitin beads. Chitin Beads should be stored at 4°C. Temporary storage at -20°C will not affect the binding capacity.
- E. coli strain ER2566 is included with all vectors sold separately. A competent version, T7 Express Competent E. coli (NEB #C2566H), can be purchased separately from NEB.
FAQs
- Where can I find many more detailed FAQs for IMPACT systems?
- What factors contribute to the low expression of some proteins when using IMPACT?
- What are some suggestions for expressing a toxic gene using the IMPACT system?
- How can I use IMPACT with an insoluble fusion protein?
- What are the advantages of the IMPACT System?
- What is the binding capacity of the resin in the IMPACT system?
- If the fusion protein forms inclusion bodies, can I try to purify protein under denaturing conditions using IMPACT?
- Can the chitin beads in the IMPACT kit be regenerated?
- What is the success rate of procaryotic protein purification using the IMPACT system?
- What size range of proteins have been purified with the IMPACT system?
References
- Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H. and Xu, M.-Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene, 192, 277-281.
- Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q. and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res., 26, 5109-5115.
- Evans, T.C., Benner, J. and Xu, M.-Q. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci., 7, 2256-2264.
Reagents Sold Separately
Anti-Chitin Binding Domain Serum
Chitin Beads
Intein Forward Primer (24-mer)
Intein Reverse Primer (24-mer)
pMYB5 Control Plasmid
pTYB1 Vector
pTYB11 Vector
pTYB12 Vector
pTYB2 Vector
T7 Terminator Reverse Primer (19-mer)
T7 Universal Primer (20-mer)
Companion Products
Anti-CBD Monoclonal Antibody
Bio-P1 (Lyophilized)
Chitin Magnetic Beads
Flu-P1 (Lyophilized)
pKYB1 Vector
pMXB10 Control Plasmid
pTWIN-MBP1 Vector
pTWIN1 Vector
pTWIN2 Vector
pTXB1 Vector
pTXB3 Vector
pTYB3 Vector
pTYB4 Vector
T7 Express Competent E. coli (High Efficiency)
Legal
Research Use Assurance:
The buyer and user have a non-exclusive sub-license to use this system for 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 explicitly prohibited. This limitation applies to E. coli strain ER2566 which is provided in combination with IMPACT™-TWIN System or in combination with appropriate vectors for said system.
Licenses/Patents/Disclaimers:
Notice to Buyer/User: The buyer/user has a non-exclusive license to use this system or any components thereof for Research Purposes Only. See Research Use Assurance Statement for details on terms of the license granted hereunder.
Information presented herein is accurate and reliable to the best of our knowledge and belief, but it is not guaranteed to be so. Nothing herein is to be construed as recommending any practice or any product in violation of any patent or violation of any law or regulation. It is the user’s responsibility to determine for himself or herself the suitability of any material and/or procedure for a specific purpose and to adopt such safety precautions as may be necessary.
A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.:
Commercial Laboratory Buyer/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™-TWIN 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; Tel. 631-344-7134, Fax: 631-344-3729.
You may refuse this non-exclusive research license agreement by returning the enclosed materials unused. By keeping or using the enclosed materials, you agree to be bound by the terms of this sub-license.
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