IMPACT™-CN System From New England Biolabs

The IMPACT™-CN protein expression and purification system is the first to utilize protein splicing-based removal of the affinity purification tag through a thiol-induced self-cleavage of the intein fused to the target protein. A set of four E. coli expression vectors included with the kit allow for creation of N- or C-terminal fusions of the target polypeptide to the affinity tag: the chitin-binding domain from Bacillus circulans, and the intein from Saccharomyces cerevisiae. Vectors pTYB1 and pTYB11 allow production of target proteins without any extra amino acids after completion of cleavage, while proteins expressed from pTYB2 and pTYB12 contain 1-3 extra amino acids depending on the choice of the cloning site.

The chitin-binding tag utilized in the IMPACT™-CN system is particularly well suited for single-column purification of proteins produced in E. coli, as the host appears to lack any endogenous proteins binding to chitin. This is in stark contrast to the popular metal-affinity hexahistidine tag, preparations of which are invariably contaminated by endogenous metal-binding proteins. The binding of the IMPACT™-CN tag to the chitin beads appears to tolerate high salt (up to 1 M NaCl) and detergents, but unlike binding of hexahistidine to Ni++ or Co++, is destroyed by denaturants. By the virtue of its purification and cleavage scheme, IMPACT™-CN expressed proteins can only be purified in non-denaturing conditions. We have used this system to produce over 20 different bacterial proteins (sizes 11-63 KDa) for crystallization and/or in vitro analysis.

In contrast to other affinity purification protocols the fusion protein is not eluted off the resin in a classical sense; instead, it is cleaved in two by the action of intein while on the column. The resulting intein-chitin-binding domain fusion remains bound to the resin, while the cleaved-off target polypeptide/protein can be eluted in any buffer. Compared to other protocols of proteolytic tag removal (e.g. using thrombin or TEV protease), the IMPACT™-CN system utilizes intrinsic "protease" activity with no need for a separate step to remove the protease or the tag. (Use of N-terminal fusions creates a small, 1.6 kDa, extein oligopeptide which co-elutes with the target; due to its miniscule size the extein is readily removed during dialysis steps).

Expression from the pTYB series of plasmids is driven by T7 RNA polymerase necessitating the use of a lambdaDE3 host; we have used BL21 derivatives for conventional expression and B834 strain for Se-Met labeling of proteins. The cells have to be disrupted largely by mechanical means (French press, sonication) since lysozyme will degrade the chitin beads. Cleared cell extract can be loaded onto the gravity column or, alternatively, the binding can be done in batch. The binding capacity and flow rate of the chitin beads vary substantially from batch to batch; occasionally the beads exhibit gel-filtration properties by retarding elution of the small proteins.

Intein-mediated cleavage is induced by prolonged incubation of the protein immobilized on beads with 50 mM beta-mercaptoethanol or DTT at 4-20ºC. Because the cleavage is triggered by thiol groups, neither compound is recommended for traditional inclusion in lysis or wash buffers as reducing agents; if reducing conditions are critical during purification, non-thiol agents such as TCEP can be used instead. Cleavage efficiency varies depending on the position and context of the intein splicing site, but usually is between 20 and 60%. Concerns have been raised regarding potential microvariability of the cleavage site; however, in my experience this microvariability, if it exists, did not affect crystallization potential or in vitro activity of the produced proteins.