pTWIN2 Vector

  • This product was discontinued on 11/01/2012
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pTWIN2 is an E. coli expression vector which can be used with the IMPACT™ Kit (NEB #E6901) (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.

Advantages and Features


  • 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., T7 Express Competent E. coli (High Efficiency) (NEB #C2566), BL21(DE3) Competent E. coli ( NEB #C2527) 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.
  • 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 acid sequence CRA or GRA at the N-terminus of the target protein is recommended for use with this intein.
  • Ampicillin resistance.

Properties and Usage

Affinity Tag

Chitin-Binding Domain (CBD)

Storage Temperature



  1. Cell Lysis Buffer: 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl.
    Ssp DnaB Intein Cleavage Buffer: 50 mM Tris-HCl (pH 7.0) containing 500 mM NaCl.
    Mth RIR1 Intein Cleavage Buffer: 50 mM Tris-HCl (pH 8.5) containing 500 mM NaCl and 50 mM 2-mercaptoethanesulfonic acid.


  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 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.


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Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.