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)].
Figure 1: Purification of Maltose Binding Protein (MBP) in a single affinity purification step Lane 1: uninduced cell extract. Lane 2: induced cell extract showing expressed fusion protein. Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C. Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703).
Figure 2: Schematic of the IMPACT System
Figure 3: Intein-mediated Protein Ligation (IPL)
Figure 4: Polylinkers in the vectors pTXB1 and pTYB21
▼ indicates intein cleavage site.
Figure 5: Flow chart for Protein Expression and Purification using the IMPACT System Sample collection for analysis by SDS-PAGE is indicated.
This product is related to the following categories:
Mauris, J.and Evans, T.C., Jr. (2010) A human PMS2 homologue from Aquifex aeolicus stimulates an ATP-dependent DNA helicase. J Biol Chem; 285(15), 11087-11092. PubMedID: 20129926
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