How the CL7 Tag Achieves Ultra-High-Affinity Binding in Protein Purification

The CL7 affinity tag, proprietary to TriAltus Bioscience, is engineered for superior ligand binding strength and specificity in protein purification workflows. This article explores how CL7’s structural design, its interaction with Im7 resin, and its compatibility with high-salt purification conditions outperform traditional tags such as His and Strep—resulting in higher purity, yield, and reproducibility across a range of protein targets and production settings.

Why Binding Strength Matters

Affinity purification relies on the selective interaction between a tagged recombinant protein and a ligand on the purification resin. The strength of this interaction dictates how effectively the protein of interest is captured and how cleanly it can be isolated from host cell contaminants.

In systems with weaker binding affinities—such as His-tag and metal ion chromatography—non-specific interactions often persist. These contaminants may co-elute, require additional purification steps, or reduce functional yield. Moreover, when purification buffers vary in salt or pH, these weak interactions may fail altogether, leading to target loss or inconsistent batch performance.

What Is CL7? A Purpose-Built Affinity Tag

The CL7 tag is a small, stable peptide engineered from the DNase domain of colicin E7, a bacterial toxin. Its compact size (~16 kDa), solubility, and non-toxic behavior make it an excellent fusion tag for recombinant expression in a variety of systems including E. coli, insect, and mammalian hosts.

Unlike other affinity tags that rely on weak ionic or metal interactions, CL7 is designed for a single, high-affinity interaction with its binding partner: the Im7 ligand. This pairing forms the basis of the CLīM™ system, TriAltus’ one-step purification platform.

The CL7–Im7 Interaction: Near-Covalent Strength

The hallmark of the CL7 system is its femtomolar-range binding affinity to Im7—orders of magnitude stronger than traditional His-tags (micromolar to millimolar) or Strep-tag systems. This interaction mimics a lock-and-key mechanism with molecular precision.

Such strength offers clear benefits:

  • Minimal non-specific binding, even in crude lysates

  • Resilience to wash buffers with high salt or detergents, allowing more aggressive removal of contaminants

  • Exceptional reproducibility, especially in applications that require consistency across replicates or scaled production

Together, these features make the CL7–Im7 interaction a powerful alternative to standard affinity systems.

Salt Tolerance = Cleaner Loads, Fewer Steps

The Im7 resin is uniquely salt-tolerant, maintaining its binding capacity even in loading conditions ranging from 0.5 to 2 M NaCl, and washing conditions up to 4M NaCl. This allows proteins to be loaded directly from clarified lysates without desalting or buffer exchange—saving time and preserving sample integrity.

In contrast, Strep and His systems often require low-salt conditions during the loading phase to prevent disruption of weak binding interactions. These limitations can allow contaminants like nucleic acids, lipids, and host proteins to remain bound to the column, reducing purity.

By permitting high-salt loading, the CL7–Im7 system enables early removal of these impurities, resulting in cleaner elutions with fewer steps.

Application Spotlight: CRISPR Tools, Structural Biology, and Multi-Subunit Complexes

High-affinity purification is particularly valuable in workflows involving:

  • CRISPR-associated proteins (e.g., Cas9, Cas12a), where trace contaminants may interfere with gene editing efficiency

  • Membrane-bound proteins, which often aggregate or bind non-specifically without robust purification methods
    Multi-subunit enzyme complexes, where stoichiometric balance is essential for function

  • Structural biology pipelines, including cryo-EM and crystallography, which demand exceptional homogeneity and purity

TriAltus customers in both academic and commercial labs have successfully used CL7-tagged systems to streamline these complex applications.

An Affinity You Can Count On
 TriAltus customers don’t need to adopt a new platform to benefit from CLīM™—they get all the advantages through our protein production services. Powered by the ultra-high-affinity CL7/Im7 system, our process delivers exceptional purity, salt tolerance, and consistency—so you receive high-performance proteins ready for genome editing, assay development, or therapeutic research.

From R&D-scale batches to custom production, TriAltus helps you move faster, cleaner, and with greater confidence—no in-house optimization required.


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