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.
The protease used depends on the configuration of the expression construct. We offer vectors that can place the CL7 tag at either the N or C terminus, with a variety of expression tags. Expression tags may need to be cleaved in addition to the CL7 tag. We use Prescission protease or SUMO protease. Our pricing for either protease is identical: $100/1mg, with price dropping to as low as $40/mg for 100 mg.
No, the only way to get your target protein is to cut it off with protease, or to elute with guanidine (or glycine). But 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. Because the protease only needs to be added at ~1.5-3.0% of the expected yield of the bound target protein, the protease may not need to be removed, depending on the final purity required.
If the protease must be removed, a column in tandem to the Im7 column can be used. PreScission protease has a GST tag and may be removed using a glutathione column; SUMO protease has a His-Tag and may be removed using a nickel column. When needed, we add this protease-removal column in sequence with the main column, so as to collect the target protein in a single chromatography step and avoid additional dilution/concentration. Alternatively, if the cleaved target protein is sufficiently different in size from the proteases, a size exclusion step may be used.
SUMO protease recognizes SUMO tertiary structure – not a specific amino acid sequence – to cleave and release the target protein from the Im7-bound CL7 tag. It cleaves after the C-terminal glycine at the end of the SUMO sequence. If the target protein is fused downstream of the SUMO C-terminus, SUMO protease cleavage renders the native protein sequence without any additional amino acids.
Prescission protease (PSC) recognizes the sequence “LEVLFQ/GP” and cleaves between the glutamine (Q) and glycine (G) residues. Our vectors include a few additional residues before/after the PSC site to allow for better access of PSC to it. Hence, using PSC to cleave an N-terminal tag will leave 2 amino acids (GP), while cleaving a C-terminal tag will leave 6 amino acids (LEVLFQ).
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.
For membrane proteins, plasmids 9 & 10 are good to try with the engineered signal peptide. Multisubunit may work well with plasmid 1. Large or multimeric proteins may do well with one of the 30’s (Plasmid 31, 32, 33, or 34), since it has a long spacer arm with multiple protease sites to maximize chances of successful cleavage. After that it’s a matter of which end you want to tag, whether they want to double tag with His, and whether they want to try an expression tag (e.g. TRX, MBP, SUMO).
Although we have not specifically tested a mammalian or insect system there is no reason that the tag sequence could not work in a mammalian vector as well. We are working on developing into mammalian and insect systems.
We have tried TEV as well as PreScission Protease and SUMO Protease, and all work fine, so we would expect other cleavage sites to work as well. You may need to engineer extra residues as a spacer between the cleavage site and protein of interest or the CL7 tag to avoid steric hindrance, but this would be determined empirically.