CL7/Im7 Tag Technology
The CL7/Im7 Tag System
The CL7/Im7 Protein Purification System is based on the ultra-high affinity complex formed between two small proteins: CL7 and Im7. CL7 (16kDa) is the tag fused to the target protein, and Im7 (10 kDa) is the ligand coupled to agarose resin.
The WT version of CL7 is CE7 DNase, a toxic bacterial protein. Mutations were engineered to abolish its DNA-binding and DNAse activities while retaining its ultra-high affinity to Im7.
The CL7 tag can be expressed at the N or C terminus of the target protein. Our plasmids offer combinations of solubility tags, affinity tags, and cleavage sites with multiple cloning options.
CL7 showed no negative effect on solubility or expression in control experiments. In fact, CL7 improves expression levels and solubility of proteins originally insoluble when expressed in E. coli with no tag. Several clinically and therapeutically relevant proteins moved from the insoluble to the soluble fraction when expressed with a CL7 tag. No denaturants or refolding from inclusion bodies was required to purify the solubly-expressed proteins.
Im7 resin is composed of CL7’s binding partner, Im7, immobilized to agarose resin. With a highly specific affinity between Im7 and CL7, off-target resin binding is minimal. With a high resin binding capacity of 35-40 mg/mL, large amounts of CL7-tagged protein can be captured. The Im7 domain is highly resilient, and the resin can be regenerated and reused up to 100 times using Gdn-HCl.
Proteases are essential for eluting the target protein off the Im7 resin column.
TriAltus purifies our own SUMO and PSC proteases which, because of their ultra-high purity, are highly active. Both proteases offered by TriAltus cleave faster and are cheaper than competitors' offerings.
For example, if 60-80 mg of protein is bound to a 5 mL column, 1 mg of protease in 20 mL of elution buffer with a flow rate of 0.2 mL/min will elute the protein in only 1.5-2 hours. The small amount of protease is so dilute that it maynot need to be removed. However, polishing steps can be performed using a His column for SUMO and GST for PSC, if desired.
SUMO protease is used to elute N-terminal tagged proteins. It is a highly specific enzyme that recognizes the tertiary structure of the SUMO cleavage site and leaves no amino acid residues after cleavage. Its specificity also allows it to cleave faster than PSC. 1 ug of SUMO cleaves 2 mg of substrate by 96% in 30 minutes and 99% in 40 minutes.
PSC protease is used to cleave N- or C- terminal tagged proteins. It is also highly efficient: 20 ug of PSC will cleave 2 mg substrate at 91% in 40 minutes and 99% in 60 minutes.
Simple purification protocol for 1 chromatography step
CL7 and Im7's salt independent and highly specific binding affinity to each other allows for a straightforward and streamlined protocol. Instead of using His-trap as an initial step and then a specialty tag to refine the protein, CL7 achieves ultra-high purity in one chromatography step.
Every protein purification system on the market can be characterized by its performance under two parameters: yield and purity. The CL7/Im7 system outperforms His-tag and specialty tags on both fronts.
TriAltus has developed a highly efficient coupling method for a high binding capacity resin. Binding capacity for Im7 resin is in the 35-40 mg/mL range. This is far superior to the resin of specialty tags and on the higher end of Ni columns for His-trap.
Purity is determined by the sensitivity of the protein purification system to untagged cellular components. There are two categories of impurities: those based on tagged target protein/cellular component interactions and those due to nonspecific binding of cellular components to the column-bound ligand.
Interactions with target protein
The binding of CL7 to Im7 is undisturbed in the presence of high salt concentration buffers. Higher salt buffers are effective at removing impurities early during purification for a clean final product. Specialty tags are sensitive to salt concentrations of about 0.2-0.3 M, leading to low purity after the first chromatography step.
CL7 and Im7 are highly specific to only each other. This prevents nonspecific interactions between the tag or ligand and cellular components such as DNA and other proteins. IMAC systems are susceptible to nonspecific binding to the metal ions in the resin.
The combination of these two factors, high salt tolerance and highly specific binding, results in such high purity that the CL7/Im7 system requires only one chromatography step.
Success purifying challenging proteins
Multi-subunit RNA Polymerase- ttRNAP
ttRNAP (Thermus Thermophilus RNA polymerase) is a large ~400 kDa protein with 5 subunits (α2ββ’ω) and 4 binding sites. Traditionally, purification requires 5 successive chromatography steps to achieve high enough purity for high activity. Purity of the protein is directly linked to its activity. Loading the cell lysate in 1.5 M salt buffer is key to one-step purity. The CL7 tag is attached to the largest β’ subunit and does not interfere with subunit assembly.
DNA/RNA-binding protein- Cas9
Cas9 is a crucial component of CRISPR technology. Cas9 activity is directly correlated to its purity but usually takes 4-5 chromatography steps to achieve desirable purity. When Cas9 is CL7-tagged and loaded in 1.5 M salt buffer, 99% purity can be achieved in one purification step. The enzyme shows high activity in both in vivo and in vitro assays.
Membrane protein- YidC
YidC is a ~32 kDa bacterial membrane integrase. Membrane proteins are notoriously difficult to purify because of contamination due to hydrophobic contacts with cellular components. Because His-tag binds non-specifically to its column, a significant amount of impurities are present after one step of purification. CL7's specificity to Im7 minimizes these effects, bringing purity up to 99%.
Poorly folded therapeutic proteins- GCSF, hGH, and IFN-α
GCSF, hGH, and IFN-α are three FDA-approved biologics. Each has two internal cysteine bridges and are usually insoluble when expressed in E. coli without tags. Purifying insoluble proteins usually involves a tricky refolding step followed by multiple chromatographic steps. CL7 enhances the expression, stability, and folding of the proteins such that they are no longer expressed as inclusion bodies.