• My NEB
  • Print
  • PDF
  • pTWIN1 Vector

    Description

    pTWIN1 is an E. coli expression vector which can be used with the IMPACT™ Kit (NEB #E6901). 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.

    DNASU is a central repository for plasmid clones and collections that may also be helpful.

    Advantages and Features

    Features

    • 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 Competent E. coli (High Efficiency) (NEB #C2566) or BL21(DE3) Competent E. coli (NEB #C2527) 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

    Properties and Usage

    Affinity Tag

    Chitin-Binding Domain (CBD)

    Storage Temperature

    -20°C

    Notes

    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 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

    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. (1998). Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci.. 7, 2256-2264.
    4. 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.
    5. 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.
    6. 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.
    7. 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.
    8. 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.
    9. 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, J. Mol. Biol..

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What is IMPACT?

    Selection Tools

    Interactive Tools

    Application Notes