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home > Product Catalogue > New England Biolabs > Nucleic Acids > IMPACT Vectors > pTWIN2 Vector
pTWIN2 Vector
Catalog # Size Concentration Price(£) Qty
N6952S 10 μg 200 μg/ml 72.00
Download:MSDS PDF


Description:
pTWIN2 is an E. coli expression vector which can be used with the IMPACT™-TWIN System (NEB #E6950S) (1). A polylinker in the vector is designed for the in-frame fusion of a target gene between the modified Ssp DnaB (2) and Mth RIR1 inteins (3). The presence of the chitin binding domain from Bacillus circulans (4,5) facilitates purification. 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). The double-stranded vector is 7192 base pairs in length.

Source:
pTWIN2 contains two mini-inteins, one derived from the Synechocystis sp DnaB intein (154 amino acids) (6) and the other from the Methanobacterium thermoautotrophicum intein (134 amino acids) (7).

Advantages:
  • A pBR322 derivative.
  • The SapI sites are recommended 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., ER2566, BL21(DE3) and derivatives].
  • Origin of DNA replication from the bacterio-phage M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid.
  • Thiol-induced cleavage of the Mth RIR1 intein is dependent on the amino acids adjacent to the intein. The amino acid Gly 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 acids CRA or GRA at the N-terminus of the target protein is recommended for use with this intein.
  • Ampicillin resistance.
  • Mth RIR1 Intein Reverse Primer (NEB #S1270S) 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:
  1. Cell Lysis Buffer: 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl.
  2. Ssp DnaB Intein Cleavage Buffer: 50 mM Tris-HCl (pH 7.0) containing 500 mM NaCl.
  3. Mth RIR1 Intein Cleavage Buffer: 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl and 50 mM 2-mercaptoethanesulfonic acid.
  4. E. coli strain ER2566 is only available to purchasers of the IMPACT™-TWIN System or replacement vectors.





References


  1. 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.
  2. 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.
  3. Evans, T.C., Benner, J. and Xu, M.-Q. (1999) The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum. J. Biol. Chem., 274, 3923-3926.
  4. 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.
  5. 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.
  6. 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.
  7. Smith, D.R. et al. (1997) Complete genome sequence of Methanobacterium thermoautotrophicum ΔH: functional analysis and comparative genomics. J. Bacteriol., 179, 7135-7155.
  8. 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


pTWIN-MBP1 Vector
pTWIN1 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.