The IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements, termed inteins, to separate the target protein from the affinity tag
- Distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step, a native recombinant protein without the use of a protease
- Able to produce a target protein without vector–derived amino acids
- Fusion to either C-terminus or N-terminus of target protein
- Isolation of proteins with or without an N-terminal methionine residue
- Ligation and labeling of recombinant proteins
- T7 promoter-driven system to achieve high levels of expression and tight transcriptional control in E. coli
The IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification strategy that utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag (1). It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step, a native recombinant protein without the use of a protease. Each intein tag contains a chitin binding domain (CBD) for the affinity purification of the fusion protein on chitin resin (2–4). Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the target protein from the intein tag (Figures 1,2). The vectors included in this kit allow for the fusion of the target protein at its C-terminus (pTXB1) (3,5) or at its N-terminus (pTYB21) (4,6) to the intein tag.
In addition, with the use of pTXB1, native recombinant proteins that possess a reactive C-terminal thioester can be isolated for applications, including protein semisynthesis and site-specific labeling [3,7, Intein Mediated Protein Ligation (IPL, Appendix I)].
The following reagents are supplied with this product:
Store at (°C) Concentration 1M DTT -20 1000 mM Blue Loading Buffer Pack -20 3 X Anti-CBD Monoclonal Antibody -20 pTXB1 Vector -20 200 µg/ml pTYB21 Vector -20 200 µg/ml pMXB10 Control Plasmid -20 0.2 mg/ml Chitin Resin 4
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