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.

    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

Affinity Tag

Chitin-Binding Domain (CBD)

Storage Temperature

-20°C