IMPACT CitationsThe IMPACT (Intein Mediated Purification with an Affinity Chitin-bindingTag) system is a novel protein purification strategy that utilizes the inducibleself-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag (1). It distinguishes itself from allother purification systems by its ability to purify, in a single chromatographicstep, a native recombinant protein without the use of a protease. Each inteintag contains a chitin binding domain (CBD) for the affinity purification of thefusion protein on chitin resin (2–4). Induction of on-column cleavage, usingthiol reagents such as dithiothreitol (DTT), releases the target protein fromthe intein tag (Figures 1,2). The vectors included in this kit allow for the fusionof 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 areactive C-terminal thioester can be isolated for applications, including proteinsemisynthesis 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 stepLane 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 SystemFigure 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 SystemSample collection for analysis by SDS-PAGE is indicated.
This product is related to the following categories:
IMPACT System,
Bacterial E. coli Protein Expression,
Chitin Purification (CBD-tag),
Affinity Purification,
Protein Purification,
Protein Expression,
Protein Labeling Products
This product can be used in the following applications:
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