CLīM™ Technology
CLīM™ Affinity Tag System
The CLīM™ Affinity Tag System is based on the ultra-high affinity complex formed between two small proteins. CL7 (16 kDa) is the tag fused to the target protein and lm7 (10 KDa) is the ligand coupled to the agarose resin.
The wildtype version of CL7 is CE7 DNase, a toxic bacterial protein. Engineered mutations abolish its DNA-binding and DNase activities while preserving ultra-high affinity to lm7.
Components
CL7 tag
The CL7 tag can be expressed at the N or C terminus of the target protein.
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. These proteins demonstrated biological activities equivalent to that of other commercial/clinical samples in cell-based assays.
Im7 resin
lm7 resin is composed of CL7's binding partner, lm7, immobilized to agarose resin. The highly specific affinity between lm7 and CL7 results in minimal off-target resin binding. The high resin binding capacity of 35-40 mg/mL allows for large amounts of CL7-tagged protein to be captured.
Elution proteases
Proteases are used for eluting the target protein from the lm7 resin column. TriAltus produces highly active, ultra-high purity SUMO and PSC proteases. Both TriAltus-purified proteases cleave faster than competitors' offerings at a lower cost.
The relatively small amount of protease used for elution is so dilute that the protease may not need to be removed from the final product. However, to remove residual protease from the final sample, an affinity column can be placed in tandem with the Im7 column to capture the tagged protease while allowing the tag-free target protein to pass through for collection.
SUMO protease is used to elute N-terminal tagged proteins. This highly specific enzyme recognizes the tertiary structure of the SUMO domain and cleaves the C-terminal to it, leaving no amino acid residues after cleavage. 1 μg of SUMO cleaves 2 mg of substrate by 96% in 30 minutes and 99% in 40 minutes at 4°C.
PSC
PSC protease is used to cleave N- or C- terminal tagged proteins. It is also highly efficient: 20 μg of PSC will cleave 2 mg substrate at 91% in 40 minutes and 99% in 60 minutes at 4°C.
One step chromatography purification protocol
CL7 and Im7's salt-independent and highly specific binding affinity 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.
Advantages
Protein purification systems are evaluated based on their performance under two parameters: yield and purity. CLīM™ Affinity Tag Systems outperforms His-tag and specialty tags on both accounts.
Yield
TriAltus' highly efficient coupling method for a high binding capacity resin is far superior to the the resin of other specialty tags and is on the higher end of Ni columns for His-trap. Binding capacity for lm7 6B resin is in the 35-40 mg/mL range and >60 mg/mL for the 4B resin.
Purity
A protein purification system's achievable purity is determined by its sensitivity to untagged cellular components. Types of impurities include those based on tagged target protein/cellular component interactions and those due to nonspecific binding of untagged 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 remove impurities early in the purification process 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.
Nonspecific binding
CL7 and Im7 are highly specific exclusively to one another. 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 high salt tolerance and highly specific binding results in such high purity that the CL7/lm7 system requires only one chromatography step.
Challenging proteins, purified
Multi-subunit RNA Polymerase- ttRNAP
ttRNAP (Thermus Thermophilus RNA polymerase) is a large ~400 kDa protein with five subunits (α2ββ’ω) and four binding sites .Traditionally, purification requires five successive chromatography steps to achieve high enough purity for high activity. Protein purity directly correlates with its activity. Loading the cell lysate in 1.5 M salt buffer is key to one-step purity. The CL7 tag is attached only 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. Cas9 that is CL7-tagged and loaded in 1.5 M salt buffer achieves 99% purity in one step purification. The enzyme shows high activity in both cell based 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, significant 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
Three FDA-approved biologics --GCSF, hGH, and IFN-α--have two internal cysteine bridges each and are usually insoluble when expressed in E. coli without tags. Purifying insoluble proteins often 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 in inclusion bodies. These proteins demonstrated biological activities equivalent to that of other commercial/clinical samples in cell-based arrays.
