|Products||IMPACT-CN System from New England Biolabs|
|Company||New England Biolabs|
|Description|| IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) utilizes the inducible self-cleavage activity of engineered protein splicing elements (termed inteins) to purify recombinant proteins by a single affinity column (1-4) (Figures 1 and 2). This system distinguishes itself from other protein fusion systems by its ability to separate a recombinant protein from the affinity tag without the use of a protease.
The IMPACT-CN Protein Fusion and Purification System contains four expression vectors (pTYB vectors) which allow fusion of a bifunctional tag, consisting of the intein and the chitin binding domain (5), to either the C-terminus (pTYB1,2) or N-terminus (pTYB11,12) of the target protein. In the presence of thiols such as DTT, -mercaptoethanol or cysteine, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag.
Both pTYB1 and pTYB11 contain a Sap I cloning site which allows the target gene to be cloned immediately adjacent to the cleavage site of the intein tag; this results in the purification of a target protein without any extra non-native residues attached to its terminus after cleavage. Use of pTYB2 or pTYB12 yields a target protein with extra residue(s) added to its C-terminus or N-terminus, respectively. Addition of extra residues may help the cleavage reaction for some target proteins. pTYB2 and pTYB12 contain the same or compatible restriction sites in the multiple cloning region. This allows cloning the same target gene fragment into both vectors. pTYB1 and pTYB2 use ATG of the Nde I site in the multiple cloning region for translation initiation.
|Info|| New England Biolabs|
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