How to circumvent common problems associated with the His-tag system?
The His-tag (6xHis-tag, His6-tag or polyhistine-tag) is a popular affinity tag for protein purification since its small size ensures a low impact on protein structure. While the His-tag system is popular for its high yield and low costs, the time and effort it takes to establish successful His-tag purification is often neglected.
- Low target purity due to unspecific binding of host proteins (Figure 1A)
- Sample or buffer components interfere with protein binding
- Protein precipitation after purification due to incompatible buffer components
- Culture media for mammalian cells interfere with protein binding or cause Ni2+ leakage
- Required pH for target protein causes elution from Ni-NTA agarose
- Limited applicability in protein analysis due to low tag-ligand affinity and specificity
To avoid or address these problems, careful planning of the procedure and optimization of the His-tag purification protocol is required. In the end, a His-tag might still not achieve satisfactory results or might not be suitable at all for a certain type of protein. In this case, a different affinity tag should be considered.
The Strep-tag® system is the ideal alternative to His-tag since all common problems are avoided. Due to the specific tag-ligand interaction, protein isolation using this system leads to highly pure protein without further optimization or processing steps (Figure 1B).
Figure 1: GFP fused to either a 6xHis-tag (A) or to a Twin-Strep-tag® (B) was isolated from E. coli lysates using standard protocols provided by the manufacturers for the used resins (Ni-NTA from Thermo Fisher Scientific for His-tag and Strep-Tactin®XT 4Flow® high capacity from IBA Lifesciences for Twin-Strep-tag®)
Watch our free on-demand about common problems during His-tag purification and read our white paper comparing His-tag and Strep-tag® protein purification systems.
What are causes of impurities during His-tag purification and how to eliminate contaminants?
His-tag is bound by transition metal ions, of which nickel (Ni2+) is most commonly used. Nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) usually serve as carrier matrices for Ni2+. Within the His-tag purification system, which is a type of immobilized metal affinity chromatography (IMAC), both the type of tag and the corresponding tag-binding metal ions (e.g. Ni2+, cobalt (Co2+) or copper (Cu2+)), potentially cause impurities:
Figure 2: Since His-containing proteins non-specifically bind to Ni-NTA resins, titration of wash and elution buffers are necessary. In addition, further processing steps such as e.g. size exclusion chromatography or filtration can be required to increase the purity sufficiently for further experiments. During this step, protein is lost, reducing the overall protein yield. In contrast, Strep-tag®II and Twin-Strep-tag® bind to their ligands with a high specificity, resulting in a highly pure protein preparation that can directly be used for further downstream applications.
- “His” or “Histidine” is an amino acid that may be present in host proteins. Consecutive His-residues mediate unspecific binding to the chelated metals on the His-tag purification resin.
- Host proteins may have an intrinsic affinity to Ni2+ or Co2+.
To increase the purity of His-tag proteins, several steps before, during and after the purification process offer optimization potential.
As a first step, elution agent imidazole can be added in a low concentration to the cell lysis buffer and to wash buffers. This way, unspecific binding of intrinsic proteins with His-tag can be prevented. However, the imidazole concentration that inhibits only the binding of His-containing host proteins and still allows the binding of 6xHis-tagged target proteins has to be carefully titrated. Similarly, gradually adjusting the imidazole concentration during elution can help to eliminate His-tag contaminants. If these approaches are not successful, increasing the number of His-residues from e.g. 6 to 10 achieves stronger binding of the target protein and permits higher imidazole concentrations during washing to remove contaminants. However, this means starting from scratch again, since protein expression has to be adapted.
If the purity of the His-tag target protein is not satisfactory after modifying the purification procedure, additional processing steps can be considered. These include size exclusion or ion exchange chromatography, filtration or dialysis. The disadvantages here are that all extra steps can cause protein loss and additional costs (Figure 2).
In contrast, the two tags that are used in Strep-tag®-based protein purification (Strep-tag®II and Twin-Strep-tag®) were specifically engineered for use in this technology. Therefore, unlike His residues, it is not likely that they naturally occur in proteins and subsequently bind to the purification resin (Figure 3). If any impurities occur during Strep-tag® purification, this is usually caused by biotinylated proteins, which are easily and specifically blocked with avidin. Otherwise, ready-to-use wash and elution can be applied as recommended.
Figure 3: E. coli lysate was added to Ni-NTA (A) or to Strep-Tactin®XT 4Flow® high capacity (HC) resin (B). Contaminants were only observed with Ni-NTA resin due to unspecific binding of proteins containing His residue.
