Common protein tags explained - His-tag and FLAG

his-tag and flag

His-tag and FLAG are two of the most commonly used tags in affinity purification. While both systems have positive features, disadvantages such as multiple chromatography steps, low resin binding capacity, and non-specific binding can result in lower purity and yield than desired.

 

His-tag

His-tag is usually composed of either 6 or 8 histidine amino acid residues in sequence (6xHis, His8). Its small size means that it’s often not cleaved from the purified target protein, although it can be if a protease site is included in the vector. Because of the His-tag’s high resistance to high salt buffers, purity anywhere from 85-95% can generally be achieved. 

However, a high final purity may only be achieved after multiple chromatography runs. The interaction between His-tag and its Nickel-based resin is non-specific, meaning that there can be off-target protein binding and contamination. It usually takes multiple steps to further refine and purify the target protein, resulting in a loss of time, reagents, and final protein yield.

  • Pros: most common, small, high salt buffer compatible
  • Cons: non-specific binding, requires multiple steps for high purity

FLAG

FLAG tag makes several improvements upon His-tag’s capabilities. Like His, it has a short 8 amino acid sequence, which is small, hydrophilic, and unlikely to interfere with protein folding or function. If desired, FLAG can be removed with enterokinase. FLAG binds to resin crosslinked with antibodies specific to FLAG (M1, M2, M5) which is helpful for detection studies.

However, enterokinase is known to be a more promiscuous protease than SUMO or PSC. A major drawback to FLAG is that its resin has a very low binding capacity of >0.6 mg protein/mL resin. Additionally, FLAG is not compatible with high-salt buffers, and therefore more chromatography steps may be required to achieve high purity protein.

  • Pros: small, has antibodies for detection
  • Cons: potential non-specific cleavage, sensitive to high salt, very low column binding capacity, high expense

How does CL7 compare?

Where His and FLAG fall short, CL7 is the best of both worlds. Its ultra-high binding affinity for its ligand Im7 means that proteins only bind specifically to the resin. This also allows for the use of high salt buffers which remove impurities thoroughly in early loading steps. For this reason, the CL7/Im7 system requires only one chromatography step to achieve ultra-high purity protein in the range of 97-100%. 

The CL7 tag can be cleaved with any protease site that’s engineered into the vector. Using a highly efficient protease such that its final concentration in the eluted protein is 1% means that a second column run is often not necessary. TriAltus’ PSC protease has a GST tag and SUMO protease has a His-tag that allows for their removal if desired and are highly efficient. Finally, Im7 resin has a much higher binding capacity of 35-40 mg/mL, putting it far above FLAG and on par with or above His. 

  • Pros: high salt buffer compatible, one step for high purity, specific binding, specific protease cleavage for elution, high resin binding capacity