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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 WT 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. 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. These proteins demonstrated biological activities equivalent to that of other commercial/clinical samples in cell-based assays.

 

insoluble proteins

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. The lm7 domain is highly resilient: the resin can be regenerated and reused up to 100 times using Gdn-HCI.

Elution proteases

Proteases are essential for eluting the target protein off the lm7 resin column. TriAltus produces its own highly active, ultra-high affinity purity SUMO and PSC proteases. Both TriAltus proteases cleave faster than competitors' offerings at a lower cost. 

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 may not need to be removed. If desired, polishing steps can be performed using a nickel column for SUMO and glutathione for PSC. 

 SUMO 

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. Its specificity allows it to cleave faster than PSC. 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. 

Questions?

Purification protocols and FAQ are available on our website. Our blog covers everything from the purification of difficult proteins to troubleshooting tips. TriAltus welcomes the opportunity to discuss the application of our CLīM™ technology to proteins of interest to our customers. Schedule a call here to discuss your research and how CLīM™ can help. Our technical team is also reachable at info@trialtusbioscience.com to answer project-specific queries.