Optimizing your IMAC purification

Results from IMAC purifications depend both on the chelated metal ion and the chelator itself

Immobilized Metal Ion Affinity Chromatography (IMAC) utilizes the affinity of histidine, cysteine and tryptophan residues on the protein surface for metal ions immobilized by chelating ligands on the chromatographic resin. The method can be used for a number of different applications but is most frequently used for the purification of His-tagged proteins.

IMAC purification of His-tagged proteins is an efficient and convenient technique, although very high purity usually requires an additional polishing purification step. IMAC is not only selective for His-tagged proteins but to some extent also for other proteins containing histidine, cysteine or tryptophan residues. Some proteins from the expression host will bind to the IMAC resin, and constitute impurities in the His-tagged protein preparation. Although this is true for protein expressed in Escherichia coli, the problem is usually more severe when purifying His-tagged proteins expressed in eukaryotic hosts. Minor differences in surface distribution of the relevant amino acids can greatly change the affinity of a protein for different metal ions. The affinities depend on the metal ion itself, as well as the type of chelating ligand used for their immobilisation.

The typical metal ions used for IMAC often show an increase in affinity in the order Zn²⁺, Co²⁺, Ni²⁺ and Cu²⁺. The type of chelator also tends to affect the affinity of proteins for the immobilised metal ion, likely because of difference in the number of available coordination sites of the metal ion for protein binding. As an example, the nitrilotriacetic (NTA) ligand is believed to chelate Ni²⁺ ions via four coordination sites, leaving two coordination sites of the Ni²⁺ ion left for protein binding, while the iminodiacetic (IDA) ligand is believed to coordinate Ni²⁺ ions via three coordination sites leaving three sites for a stronger protein binding. Such difference in affinity, especially for untagged host cell proteins (the tentative impurities) may partly explain the differences in purification obtained with different resins. It may be added that ligand concentration and structural properties of the resin also affect the purification performance. It can be concluded that testing different IMAC resins for best purification results is highly recommended and a screening kit is a very useful tool.

The GoBio™ Mini His-tag Screening kits are available in two column sizes

The GoBio™ Mini His-tag Screening kits are available in two column sizes; 1 mL and 5 mL, prepacked with WorkBeads™ NTA and WorkBeads IDA, containing the chelating ligands mentioned earlier. The screening kits for each ligand contains ready-to-use, prepacked GoBio Mini columns, charged with four different metal ions, Ni²⁺, Co²⁺, Cu²⁺ and Zn²⁺. This diversity, combined with the excellent mechanical properties and tight size distribution of the WorkBeads resin, provides the perfect starting point to discover the optimal resin configuration for your protein.

Figure 1. Comparison of purifications of His₆-GFP on GoBio Mini NTA 1 mL and GoBio Mini IDA 1 mL charged with Ni²⁺, Co²⁺, Cu²⁺ and Zn²⁺. Blue line: absorbance at 280 nm. Red line: absorbance at 495 nm (specific for His₆-GFP). Green line: elution buffer, %.

Capacity and yield can vary considerably using seemingly very similar resins

Ni-NTA resins are the commonly recommended starting point for His-tagged protein purification since they give excellent results in most purifications. However, Co²⁺-resins has been increasingly popular since they may further improve purification in some cases. These resins often give higher purity, but usually to the cost of product yield. Therefore, testing other metal ion and chelator combinations as well as resin matrices may further improve your results.

Purification results can also vary considerably, also when working with highly concentrated target proteins at small sample volumes. As shown, the product yield can improve considerably changing to the Bio-Works product brand, without having to exchange with metal ion you use.

Figure 2. Comparison of purifications of His₆-GFP on GoBio Mini NTA 1 mL charged with Ni²⁺, Co²⁺ and other supplier’s products. Blue line: absorbance, 280 nm. Red line: absorbance, 495 nm (specific for His₆-GFP). Green line: elution buffer, %.

Table 1. Description of the yields and peaks width of each individual chromatogram presented in Figure 2.

Calls to exchange toxic metal compounds in bioprocessing

Recent times and development of IMAC for process-scale purification of biopharmaceuticals have raised concerns regarding the health and environmental issues associated with the use of toxic metal ions, such as Ni²⁺ and Co²⁺. Both are known to cause allergic reactions upon contact with the human skin as well as being carcinogenic when ingested. The inevitable leaching of ions from the resin during purification therefore makes such products questionable when processing pharmaceutical compounds.

Zn²⁺ has been found to be a possible non-toxic substitute for Ni²⁺, and can provide comparable purity, yield and elution pattern, making it a good alternative to resins based on Ni²⁺ (or Co²⁺). The similarities in functionality of the two resins is consistent between elution modes, i.e., stepwise or gradient elution, as well as protein load.

Figure 3. Comparison of purifications of His₆-GFP on GoBio Mini NTA 1 mL charged with Ni²⁺ and Zn²⁺ using different protein loads and elution modes. Blue line: absorbance, 280 nm. Red line: absorbance, 495 nm (specific for His₆-GFP). Green line: elution buffer, %.

Conclusions

• The choice of metal ion and chelator ligand of IMAC resins affect purification results
• GoBio Mini His-tag Screening kits can be used for effective screening of metal ion and chelating ligands combinations to get the best results when purifying His-tagged proteins
• Due to the health and safety issues related to using toxic metal ions in purification of pharmaceuticals as well as other process scale compounds, Zn²⁺ is an attractive non-toxic alternative to Ni²⁺-ion- and Co²⁺-ion-based IMAC for the purification of future His-tagged biopharmaceuticals