Angelo DePalma Ph.D. Writer GEN

The most popular protein tagging method has many advantages and few caveats.

Fusion tags have become indispensable for isolating pure proteins from varied sources. Proteomics and genomics studies have further underscored the importance of tagging techniques, for protein detection and solubility enhancement as well as purification.

Tagging methods that add amino acid sequences to native protein, followed by affinity purification, are numerous. For example the ID4 method adds the short hydrophilic sequence TETSQVAPA, derived from the C-terminus of bacterial rhodopsin, to target antibodies. Labeled proteins are captured using resins containing immobilized Rho 1D4 monoclonal antibody, which binds to the peptide sequence.

The poly-peptide (repeat) affinity tags poly-Arg and poly-His work similarly. The first ones employed naturally occurring poly-regions. More recently these constructs—ranging in size from six to about ten repeats—are enzymatically incorporated into proteins of interest, and usually removed after isolation.

In use since the early 1980s, poly-arginine tagging uses five or six arginine residues, each containing four electron-donating nitrogen atoms. PolyArg tagged proteins bind to cation exchangers and elute under a sodium chloride gradient at high pH.

PolyArg tagging failed to fulfill its promise due to two main shortcomings: Carboxypeptidase cleavage occurs in unacceptably low yields, and the tag itself may disrupt proteins containing a hydrophilic C-terminus.


By far the most popular protein tagging method for purification is polyhistidine tagging, or polyHis-tagging. Consisting of between three and ten histidine residues, polyHis sits at either the C- or N-terminus, is somewhat smaller than polyArg, and is less immunogenic.

Unlike polyArg, which requires an antibody-modified resin, polyHis relies on immobilized metal affinity chromatography (IMAC), which exploits the affinity of free coordination sites on the transition metal ions Cu2+, Co2+, Ni2+, and Zn2+ for electron-rich histidine residues. IMAC residues are far less expensive than antibody-modified resins, and withstand a greater number of regeneration cycles. After tagging and capture, protein-metal dissociation is accomplished through an imidazole gradient, pH alterations, or metal chelation. Regeneration of the his-tagged protein generally occurs cleanly and predictably.

Among the four preferred IMAC metal ions, the series Cu2+, Ni2+, and Zn2+, Co2+ exhibits increasing specificity but decreasing affinity. A study by Cube Biotech on a panel of three proteins purified by two nickel- and one cobalt-based purification medium showed yield reductions as high as 81% for cobalt compared with nickel.

Media for isolating his-tagged proteins include conventional resins used in column chromatography, metal-bound filtration membranes, magnetic beads, and nanoparticles. Virtually any sturdy medium capable of functionalization with an appropriate metal-containing structure is suitable.

For example, a group at the University of Michigan reported in 2011 on a method for growing poly(2-hydroxyethyl methacrylate) appendages onto magnetic nanoparticles, and functionalizing these polymer “brushes” with nitrilotriacetate-Ni2+ to produce magnetic beads that capture his-tagged proteins directly from cell extracts.

The robustness of his-tagging makes it ideal for high-throughput screening of protein expression systems, as well as for solubility studies. The most common format is the 96-well microplate, using agarose- or magnetic bead-based affinity resins in filter microplate or with plate centrifuges. With the assistance of automated liquid handlers and other components, the process may be partly or completely automated for thousands of samples.

Scientists from Qiagen have published extensively on automated processes for screening and isolating microgram quantities of various proteins, including membrane proteins and zinc-finger proteins, under both native and denaturing conditions.

Transition Metal and Ligand Choices

PolyHis tagging took nearly two decades from its invention, in 1975, and widespread adoption. The number of citations for polyHis remained flat for 20 years, then increased exponentially through the early 2000s.

Early polyHis tagging methods were based on a nickel-nitrilotriacetic acid matrix, which has been associated with nonspecific protein binding. The imidazole dissociation, moreover, induces protein aggregation.

TALON, a variation on the IMAC theme, is a cobalt-based resin specific for his-tagged proteins. Numerous vendors supply TALON resins, including BD, Wolfson, GE, Bio-Rad, Knauer, Thermo Fisher, Sigma-Aldrich, and ClonTech. Cobalt has lower affinity for polyHis than nickel, which can mean lower yields, especially from dilute samples. Cobalt-based affinity is also restricted to adjacent histidine residues, while nickel is somewhat more forgiving.

Generally, cobalt’s higher selectivity means that it binds less readily to proteins that are not specifically labeled. Depending on experimental conditions, this might result in less-vigorous washing requirements and milder elution protocols, and overall less severe conditions.

IMAC affinity matrices based on nickel are by far the most popular. The choice between nickel and cobalt-based resins (the next most popular choice) depends on many factors. When yield is important nickel is the metal of choice; where yield is immaterial or when molecules are not sensitive to elution conditions, cobalt-based affinity matrices may be more effective. Many of the vendors listed above offer both nickel- and cobalt-based affinity materials.

In a recent application note, Cube Biotech suggests, “let your protein determine your ligand and metal ion,” advising investigators to look into several binding media. While the inverse relationship between specificity and affinity for the series Cu2+, Ni2+, and Zn2+, Co2+ generally holds, many exceptions exist.

The metal’s substrate, iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) for nickel, and NTA for cobalt, differ in how they bind transition metals, which affects the nature of his-tagged protein binding.

NTA resins show less metal ion leaching than IDA, and withstand reducing and chelating agents better. By contrast IDA ligands are generally less expensive, and require a less severe imidazole gradient for elution. IDA is also smaller than NTA, so it couples to the matrix at higher density, theoretically providing more binding sites for his-tagged proteins. According to Cube Biotech optimizing purity and recovery may be possible by combining the loading density of ligands with metal affinity and specificity.

In many situations the choice comes down to experience and personal preference. Investigators who wish to test various metal chelators could investigate the full range of transition metals, for example through Vivapure microcentrifuge spin columns from Vivascience. Columns are available in membrane-immobilized nickel, cobalt, copper, and zinc, and purification takes 30 minutes. The membranes will not be identical to a lab’s IMAC medium of choice, both in terms of metal matrix and size, but they could provide some idea on the optimal IMAC metal.

Large-Scale Bioprocess Potential

Nickel-based IMAC columns scale well from milliliter to liter volumes. However, concerns over the potential immunogenicity of the polyHis sequence, and allergenic effects from nickel leaching from the purification matrix, have delayed adoption of IMAC in large-scale biomanufacturing.

Investigators have determined that given therapeutic dosing and known leaching kinetics the nickel dosage would be far below toxic levels for most processes and drugs. Enzymatic removal of polyHis is feasible in a bioprocess. This strategy has been employed for development-stage vaccines since the 1990s.

In many instances, IMAC resin manufacturers can eliminate nickel leaching, for example Roche’s cOmplete His-Tag Purification Resin. Other vendors have also claimed low leakage. The suitability of these resins to large-scale purification remains mostly unexplored.

Regardless, bioprocessors are wary of unit operations that entail regulatory risk. The most popular biotherapeutic class in terms of volume and value, monoclonal antibodies, are mostly purified through platform technologies that begin with protein A affinity capture. This is unlikely to change regardless of the efficiency of a method based on his-tagging. That leaves “other” recombinant proteins as the likely entry point for large-scale IMAC purification. Even for these low-dose products, his-tagging entails a tagging step and probably a de-tagging step.

Given the variety and broad selectivity of standard chromatography media, large-scale purification for these proteins will likely rely on IMAC only as a last resort—for labile molecules or where yields are unacceptably low.


His-tagging is a powerful technique for purifying proteins in research quantities for characterization, activity determination, proteomics studies, and with biomanufacturing in mind, the identification of high-producing cell lines. Vendors now provide numerous choices of metal ion, ligand, and affinity matrix, and all top firms amply document performance of tagging and affinity products.

His-tagging is not without its caveats. Potential changes in protein function may result, due to significant alterations in charge. This possibility may be mitigated by switching the tag from one terminus to the other, or incorporating a flexible linking moiety. Users should also avoid tagging proteins containing metals that might themselves bind to the column at a binding site lacking a coordination metal. Finally, they should exercise caution with proteins with naturally occurring histidine or cysteine residues due to non-specific binding or decreased purification efficiency.