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This product has been discontinued and replaced by IMPACT Kit (E6901)
Description:
The IMPACT™-TWIN Protein Fusion and Purification System utilizes the inducible self-cleavage activity of engineered protein splicing elements (termed inteins) for protein purification and manipulation. It can produce a target protein with an N-terminal cysteine and/or a C-terminal thioester for protein labeling, ligation and cyclization.
The System contains three expression vectors that allow the fusion of a target protein to modified mini-intein tags. These vectors permit the isolation of proteins with a C-terminal thioester for protein ligation or with an N-terminal residue other than Met (7-9). The protein ligation reaction allows the incorporation of non-coded amino acids or labels into a protein sequence (see companion products Flu-P1, NEB #P6606 and Bio-P1, NEB #P6607). Furthermore, pTWIN vectors can be used to fuse a target protein to the C-terminus of intein1, which can be induced to undergo cleavage by a temperature/pH shift (9). This is advantageous for purifying target proteins that are sensitive to reducing agents. Finally, insertion of the target protein between two intein tags allows the generation of circular protein species with a peptide bond at the site of ligation (7).
Note: The controlled cleavage activity of an intein tag can be affected by the target protein sequence. Please see IMPACT vectors and applications for amino acids that are most likely to cleave as expected. Also, when creating circular proteins it is typical that the linear form will be co-purified, requiring a further step to separate the two protein species.
Figure 1: Schematic of the IMPACT-TWIN System
Figure 2: Intein-mediated Protein Ligation (IPL)
Advantages:- Single-column purification--no additional steps to remove the affinity tag
- Isolation of proteins with or without an N-terminal methionine residue
- Use of either thiol reagent or pH and temperature shift to induce on-colum cleavage
- The only system that produces proteins possessing an N-terminal cysteine and/or C-terminal thioester for use in protein labeling, ligation and cyclization
- IPTG-inducible T7 Promoter (6) for high-level expression and tight regulation of expression
- Yields native amino acid sequence
- Flexibility of fusion to either the C-terminus or N-terminus of the target protein maximizes the probability of successful expression and purification
- Release of fusion partner without the use of proteases
- The ability to label the C-terminus of the target protein
- Allows Intein-mediated Protein Ligation (IPL)
Kit Components:
E. coli Strain ER2566
Mth RIR1 Intein Reverse Primer (18-mer)
Mxe Intein Reverse Primer (18-mer)
Ssp DnaB Intein Forward Primer (20-mer)
1, 4-Dithiothreitol (DTT)
Anti-Chitin Binding Domain Serum
Blue Loading Buffer
Chitin Beads
pTWIN-MBP1 Vector
pTWIN1 Vector
pTWIN2 Vector
Storage Conditions
Storage Temperature:
-20°C
Notes
General notes:- The controlled cleavage activity of an intein tag can be affected by the target protein sequence. Also, when creating circular proteins it is typical that the linear form will be co-purified and may require a further step to separate the two protein species.
- Sequencing Primers: 3 sequencing primers are included in the IMPACT-TWIN kit, the Ssp DnaB Intein Forward Primer (NEB#S1269S), the Mxe Intein Reverse Primer (NEB#S1268S), and the Mth RIR1 Intein Reverse Primer (NEB#S1270S). The T7 Universal Primer (NEB#S1248S) and the T7 Terminator Reverse Primer (NEB#S1271S) are sold separately. See Figure 2 for the sequencing primer positions.
- 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 their 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?
Figure 3: IMPACT-TWIN as a C-terminal intein-tag. The pTWIN vectors permit the expression of a target gene with a C-terminal intein-tag. The target protein can be purified in one-column step without the use of proteases. If 2-mercaptoethanesulfonic acid (MESNA) is used to induce intein cleavage the target protein can be ligated to a peptide or protein possessing an N-terminal cysteine. SDS-PAGE. Lane 1; induced, crude cell lysate. Lane 2; induced, crude cell lysate after passage over chitin resin. The disappearance of the induced band indicates it bound to the chitin resin. Lane 3; Chitin resin elution after MESNA-induced cleavage of the intein-tag. Lane 4; ligation of a peptide with an N-terminal cysteine to the intein purified protein. The ligation product has a slower migration than the starting reactants.
Figure 4: IMPACT-TWIN as a N-terminal intein-tag. The pTWIN vectors also permit the expression of a target gene with an N-terminal intein-tag. The target protein can be purified in one-column step without the use of proteases. A pH and temperature shift allows the purification of a protein with an N-terminal cysteine. A protein with an N-terminal cysteine can be used in ligation as illustrated in Figure 2. Protein purification using an N-terminal intein-tag. SDS-PAGE. Lane 1; induced, crude cell lysate. Lane 2; induced, crude cell lysate after application to a chitin resin. The disappearance of the induced band indicates it bound to the chitin resin. Lane 3; Purified protein with an N-terminal cysteine eluted from the chitin resin after inducing cleavage of the intein-tag.
References
- Evans, T.C., Benner, J. and Xu, M.-Q. (1999) The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins. J. Biol. Chem., 274, 18359-18363.
- Evans, T.C., Benner, J. and Xu, M.-Q. (1999) The in vitro ligation of bacterially expressed proteins using an intein from Methano-bacterium thermoautotrophicum. J. Biol. Chem., 274, 3923-3926.
- Mathys, S., Evans, T.C., Chute, I.C., Wu, H., Chong, S., Benner, J., Liu, X.-Q. and Xu, M.-Q. (1999) Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene, 231, 1-13.
- Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.-Q. and Perler, F.B. (1999) Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the Mycobacterium xenopi Gyrase A Intein. Biotechniques, 27(1), 110-121.
Reagents Sold Separately
Mth RIR1 Intein Reverse Primer (18-mer)
Mxe Intein Reverse Primer (18-mer)
Ssp DnaB Intein Forward Primer (20-mer)
Anti-Chitin Binding Domain Serum
Chitin Beads
pTWIN-MBP1 Vector
pTWIN1 Vector
pTWIN2 Vector
Companion Products
Anti-CBD Monoclonal Antibody
Bio-P1 (Lyophilized)
Chitin Magnetic Beads
Flu-P1 (Lyophilized)
IMPACT-CN System
pKYB1 Vector
pMXB10 Control Plasmid
pMYB5 Control Plasmid
pTXB1 Vector
pTXB3 Vector
pTYB1 Vector
pTYB11 Vector
pTYB12 Vector
pTYB2 Vector
pTYB3 Vector
pTYB4 Vector
T7 Express Competent E. coli (High Efficiency)
Legal
Research Use Assurance:
The buyer and use 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™-CN 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 attached hereto as Appendix V 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 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™-CN 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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