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  • FAQ: What is IMPACT?

    IMPACT, Intein-Mediated Purification with an Affinity Chitin-binding Tag, is a novel protein purification system that allows recombinant proteins to be purified without affinity tag in a single chromatographic step. This method was developed at New England Biolabs (NEB) from studies of the mechanism of protein splicing (see references in 1.10 and 1.11). It distinguishes itself from all other purification systems by its ability to purify a recombinant protein with its native sequence by a single affinity column, without the use of a protease. The IMPACT system utilizes the inducible self-cleavage activity of an engineered protein splicing element (intein) to separate the target protein from the affinity tag. The target protein is fused to a tag consisting of the intein and the chitin binding domain (CBD) which allows affinity purification of the fusion precursor on the chitin column. The intein undergoes specific cleavage by a thiol reagent or pH and temperature shift which releases the target protein from the chitin-bound tag resulting in a single column purification of the target protein.

    The IMPACT system includes a series of E. coli expression vectors, which utilize engineered inteins of 134-454 amino acid residues. These vectors are designed for protein expression and purification in E. coli as well as protein manipulations such as protein labeling, ligation and cyclization.

    The IMPACT Kit, as well as vectors sold separately, is available to meet your research goal. The compatibility of the multiple cloning sites of the vectors allows insertion of the same target gene fragment into different vectors for optimal expression and purification.

    The gene encoding the target protein is inserted into the multiple cloning site of the IMPACT expression vector, to create an in-frame fusion between the target gene and the affinity tag consisting of the intein and chitin binding domain (CBD, 52 amino acid residues) When crude extracts of induced E. coli cells are passed over a chitin column, the fusion protein of the target protein intein-CBD binds to the chitin beads while all other contaminants are washed off the column. On-column cleavage is induced at 4°C by addition of a reducing agent (such as DTT) or temperature and pH shift (pTWIN vectors). The target protein is released while the intein-CBD tag remains bound to the column, resulting in a single-column purification of the target protein.

    The IMPACT Kit contains expression vectors, which allow the fusion of the cleavable intein tag to either the C-terminus (pTXB1) or N-terminus (pTYB11) of the target protein. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein.

    The pTWIN vectors (#N6951S and #N6952S) offer many advantages: (1) the facile isolation of native proteins without the use of a thiol reagent - using pH and temperature shift (Intein 1) (2) the isolation of proteins with an N-terminal cysteine [for intein mediated ligation (IPL)] or residue other than methionine without the use of exogenous proteases which can be costly and non-specific (3) the purification of proteins with a C-terminal thioester for use in IPL reactions which can insert non-coded amino acids into a protein or label a bacterially expressed protein (4) the generation of circular protein species.

    All vectors use a T7 promoter and the lacI gene to provide stringent control of the fusion gene expression. Binding of the lac repressor to the lac operator sequence immediately downstream of the T7 promoter suppresses basal expression of the fusion gene in the absence of IPTG induction. All vectors carry the Ampr gene marker (the bla gene), which conveys ampicillin resistance to the host strain, except for pKYB1 (#N6706S) , which carries the kanamycin resistance gene.