What vectors are included in the IMPACT-TWIN kit?
The IMPACT-TWIN kit includes three E. coli expression vectors – pTWIN1, pTWIN2 and pTWIN-MBP1. The pTWIN vectors allow for the fusion of a mini-intein tag to the C-terminus, N-terminus, or both the C- and N-termini of a target protein, depending on the cloning sites chosen. Fusion of the intein tag to the C-terminus of the target protein allows thiol-dependent purification of the target protein from the chitin resin. This thiol-dependent cleavage permits the purification of proteins with a C-terminal thioester for use in intein-mediated protein ligation (IPL) reactions. IPL reactions can be used to incorporate fluorescent or biotinylated tags and/or non-coded amino acids into the C-terminus of a bacterially-expressed protein. Alternatively, fusion of the intein tag to the N-terminus of the target protein permits the release of the target protein from the chitin resin by a pH and temperature shift. This is advantageous for the purification of proteins that are sensitive to reducing agen
