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Description:
pTWIN1 is an E. coli expression vector which can be used with the IMPACT™ System. pTWIN vectors are designed for protein purification or for the isolation of proteins with an N-terminal cysteine and/or a C-terminal thioester (1). A polylinker in the vector is designed for the in-frame fusion of a target gene between the modified Ssp DnaB (2) and Mxe GyrA inteins (3,4). The presence of the chitin binding domain from Bacillus circulans (5,6) facilitates purification. The double-stranded vector is 7,375 base pairs in length.
Source:
pTWIN1 contains two mini-inteins, one derived from the Synechocystis sp DnaB intein (154 amino acids) (7) and the other from the Mycobacterium xenopi GyrA intein (198 amino acids) (8).
Advantages:- A pBR322 derivative.
- The SapI sites should be used for directional cloning of both the 5´ and 3´ ends of an insert.
- Expression of the fusion gene is under the control of the T7 promotor (8) and is regulated by IPTG due to the presence of a lacI gene.
- Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., T7 Express Cells or BL21(DE3) and derivatives] (9).
- Origin of DNA replication from the bacteriophage M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid.
- Thiol-induced cleavage of the Mxe GyrA intein is dependent on the amino acids adjacent to the intein. The amino acid residues M or Y at the C-terminus of the target protein is recommended for use with this intein.
- Controllable cleavage of the Ssp DnaB intein is dependent on the amino acids adjacent to the intein. The amino acid residues CRA or GRA at the N-terminus of the target protein is recommended for use with this intein.
- Ampicillin resistance.
- Mxe Intein Reverse II Primer (NEB #S1285) and Ssp Intein Forward Primer (NEB #S1269) are available for sequencing the insert.
Concentration:
200 μg/ml
Storage Conditions
Storage Conditions:
10 mM Tris-HCl
1 mM EDTA
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Product ER2566 is only available to purchasers of the IMPACT™ System or replacement vectors.
- Cell Lysis Buffer: 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl.
- Ssp DnaB Intein Cleavage Buffer: 50 mM Tris-HCl (pH 6.0) containing 500 mM NaCl.
- Mxe GyrA Intein Cleavage Buffer: 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl and 50 mM 2-mercaptoethanesulfonic acid.
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.
- 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.
- 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.
- 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, 271-281.
- Watanabe, T., Ito, Y., Yamada, T., Hashimoto, M., Sekine, S. and Tanaka, H. (1994) The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol., 176, 4465-4472.
- Wu, H., Xu, M.-Q. and Liu, X.-Q. (1998) Protein trans-splicing and functional mini-inteins of a cyanobacterial DnaB intein. Biochem. Biophys. Acta, 1387, 422-432.
- Telenti, A., Southworth, M., Alcaide, F., Daugelat, S., Jacobs, W.R. Jr. and Perler, F.B. (1997) The Mycobacterium xenopi GyrA protein splicing element: Characterization of a minimal intein. J. Bacteriol., 179, 6378-6382.
- Dubendorff, J.W. and Studier, F.W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol., 219, 45-59.
- Southworth, M.W., Amaya, K., Evans, J., 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, 110-120.
Companion Products
pTWIN-MBP1 Vector
pTWIN2 Vector
Legal
Research Use Assurance:
The buyer and user have a nonexclusive sublicense to use this system for any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.
Transfer of this vector in host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited.
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.
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 vector in 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; 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.
Note: E. coli strain ER2566 is only available to purchasers of the IMPACT™ System or replacement vectors.
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