Product Class: Other

pTWIN1 Vector

Product Introduction

pTWIN1 is an E. coli expression vector designed for protein purification or for the isolation of proteins with an N-terminal cysteine and/or a C-terminal thioester. A polylinker in the vector is designed for the in-frame fusion of a target gene between the modified Ssp DnaB and Mxe GyrA inteins. The presence of the chitin binding domain from Bacillus circulans facilitates purification.

  • View sequence details
  • Can be used with the IMPACT™ Protein Purification System
  • Double-stranded vector
  • 7,375 base pairs in length
  • Ampicillin resistant
Catalog # Size Concentration
N6951S 10.0 µg 200 µg/ml

Product Information

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.

This product is related to the following categories:
IMPACT System,
Bacterial E. coli Protein Expression Products,
DNA Plasmids & Substrates Products,
Protein Expression Products,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Lac Repressor on Plasmid

0

Affinity Tag

Chitin-Binding Domain (CBD)

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

Product 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 usingmodified 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 intoautocatalytic N- and C-terminal cleavage elements: facile production of proteinbuilding 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-cleavableaffinity 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..

Protocols, Manuals & Usage

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is IMPACT?
  2. What is the DNA sequence of this product?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

NEW ENGLAND BIOLABS® is a registered trademark of New England Biolabs, Inc.

U.S. Patent Nos: US 7001745 and US 7,271,256

Notice to Buyer/User: The buyer/user had a non-exclusive license for Research Purposes Only. See RESEARCH USE ASSURANCE STATEMENT on neb.com. 

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