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- Expression of the fusion gene is under the control of an IPTG -inducible T7 promoter (5).
- E. coli strains T7 Express (NEB #C2523H) or BL21 (DE3) and derivatives can be used as expression hosts.
- A pTXB1 derivative
- Ampicillin resistance
Description:
pMXB10 Control Plasmid is a control plasmid for the IMPACT™ Kit (1,2). This plasmid carries the E. coli malE gene, encoding the maltose binding protein (MBP, 42 kDa)(3), fused in-frame to the coding region of the Mxe gyrase A intein-chitin binding domain (28 kDa) from pTXB1 (2,4,5). pMXB10 can be used to test plasmid transformation, cell culture, induction and purification procedures. After induction with 0.3 mM IPTG at 30°C for 3 hours (or 15°C for 12-16 hours), 100 ml of cells should yield 2-3 mg of a 70 kDa fusion protein. After chitin column purification and cleavage, approximately 1.5-2.0 mg of MBP is usually obtained. This double stranded vector is 7813 bp in length.
Source:
pMXB10 Control Plasmid is isolated from an E. coli strain (r- m-) by a standard plasmid purification procedure.
Concentration:
0.2 mg/ml
Storage Conditions
Storage Conditions:
10 mM Tris-HCl
1 mM EDTA
pH 8.0 @ 25°C
Storage Temperature:
-20°C
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, 271-281.
- Evans, T.C., Benner, J., and Xu, M.-Q. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Prot. Sci., 7(11), 2256-2264.
- Guan, C. D. 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.
- 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.
- 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
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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