The CL7/Im7 purification system takes advantage of the ultra-high-affinity interaction between Colicin DNAse (CE7) and its inhibitor, Immunity protein 7 (Im7). For our purification system, CE7 was mutated to create a CL7 tag, which retained its full binding affinity to Im7 but was inactivated as a DNAse. The binding capacity of the CL7/Im7 purification system is 35-40 mg CL7/mL resin.
During affinity purification, CL7 tagged protein of interest binds to immobilized Im7 (cross-linked to agarose 6B beads) and can be eluted off the column with a protease. The CL7 tag has worked with a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins.
No, the only way to get your target protein is to cut it off with protease, or to elute with guanidine (or glycine). However, eluting with guanidine rather than cutting with protease denatures the target protein and leaves the CL7 tag fused on.
You may test elute with guanidine or glycine just to see if it worked, or to see how clean it is, but the target protein probably would not be active and would most likely require a refolding step. And again, it would still have the CL7 tag, which has disadvantages.
It depends on how pure you need the protein to be. Since you only need to use protease at 1.5-3% of the total amount of expected target yield, you may not need to remove it.
If you need to remove the protease (in the case of higher amounts of protease required to release the target, or because of extra-high purity requirements), you can run another column in tandem with the Im7 column. Our proteases are tagged, so you can use a GST column to remove PreScission protease, or a nickel column to remove SUMO protease.
Yes! We currently offer Starter Kit: 1-mL Gravity Flow Column – Pre-Loaded Im7 Resin. This kit consists of a 1-mL Gravity Flow Column packed with Im7 resin, all necessary buffers for purification, and your choice of one plasmid and one protease.