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Prices are in US dollars and valid only for US orders.
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Description:
pTWIN-MBP1 is a control plasmid in the IMPACT™-TWIN System (NEB #E6950S) (1). This vector has the E. coli maltose binding protein (2) fused between the modified Ssp DnaB (3) and Mxe GyrA inteins (4). The presence of the chitin binding domain from Bacillus circulans (5,6) facilitates purification. The pTWIN-MBP1 vector permits the isolation of a circular MBP species (1). The double-stranded vector is 8,518 base pairs in length.
Source:
pTWIN-MBP1 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.
- Expression of the fusion gene is under the control of the T7 promotor (9) 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., ER2566, BL21(DE3) and derivatives].
- 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.
- Ampicillin resistance.
- Mxe Intein Reverse Primer (NEB #S1268S) and Ssp DnaB Intein Forward Primer (NEB #S1269S) 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™-TWIN System or replacement vectors.
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.
- Guan, C., Li, P., Riggs, P.D. and Inouye, H. (1988) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene, 67, 21-30.
- 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. (1977) 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.
Companion Products
pTWIN1 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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