FAQ: What vectors are included in the IMPACT kit?

The IMPACT kit includes three E. coli expression vectors, which allows for the fusion of the cleavable intein tag to either the C-terminus (pTXB1) or N-terminus(pTYB11) of the target protein.

The pMXB10 vector serves as a positive control for expression and purification and can also be used as a cloning vector.

pTXB1 (#N6707S) contains a mini-intein from the Mycobacterium xenopi gyrA gene (Mxe GyrA intein; 198 amino acid residues) that has been modified to undergo thiol-induced cleavage at its N-terminus (Evans, T.C., et al. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 7, 2256–2264; Southworth, M. W., et al.(1999) BioTechniques 27, 110–120). The vector allows for the purification of a target protein without any extra amino acids by cloning into the NdeI and SapI sites. The target protein is fused at its C-terminus to a self-cleavable intein tag (~28 kD) that contains the chitin binding domain (CBD, 6 kDa) allowing for affinity purification of the fusion precursor on a chitin column. 

The pTYB11 (#N6701S) vector utilizes an intein from the Saccharomyces cerevisiae VMA1 gene (Sce VMA1 intein; 454 amino acids)(Chong, S. et al. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109–5115; Chong, S., et al. (1998) Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273, 10567–77). The target protein is fused at its N-terminus to a self-cleavable VMA1 intein-CBD tag (56 kD); the tag allows for the affinity purification of the fusion precursor on a chitin column. The vector is designed to allow for purification of a target protein without any extra amino acids, or without an N-terminal methionine residue, by cloning its 5’ end into the SapI site. 

The control vector, pMXB10 (#N6903S), derived from pTXB1 carries the control target protein, maltose-binding protein (MBP), already inserted upstream of the Mxe GyrA intein-CBD. Induction yields the MBP-GyrA intein-CBD fusion which, when cleaved, results in the elution of MBP. The polylinker regions flanking the coding region for MBP can conveniently be used to clone a gene of interest. However, after intein cleavage the target protein will contain additional amino acids at its C-terminus, including (LEY), which has had a high rate of successful cleavage. 

The IMPACT vectors utilize a T7 promoter to provide stringent control of expression of the fusion gene in E. coli (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.). The IMPACT vectors carry the Ampr gene marker (the bla gene), which conveys ampicillin resistance to the host strain; one vector, pKYB1 (#N6706S), is kanamycin resistant (available separately).