Keeping Tabs on Polyhistidine Tags

Attitudes toward Affinity Purification Range from a Desire to Move beyond Old Specificity/Yield Trade-Offs to a Willingness to Explore New Polyhistidine Technology Spin-Offs

Affinity tagging, a popular method for capturing or immobilizing proteins, has several incarnations, none more popular than polyhistidine tagging. In polyhistidine tagging, 6 to 10 histidine residues are added recombinantly to either the protein’s C- or N-terminus.

Fewer or more histidines are also possible. After expression, the protein is captured by means of immobilized metal affinity chromatography (IMAC). Subsequently, the protein is eluted in high purity by swamping the column with imidazole, EDTA, or some other strong chelator.

Histidine tagging is by no means the only affinity tagging option. Some common alternatives are maltose-binding protein and glutathione-S-transferase. Both of these tags improve fusion protein solubility and expression levels but exhibit immunogenicity for purposes of raising antiprotein antibodies. Other choices include calmodulin-binding peptide, Strep-tag (oxtapeptide binding to streptavidini), chitin-binding domain, and FLAG-tag (another octapeptide that binds to anti-FLAG mAbs).

Specificity vs. Yield

The most common affinity resins are nickel nitrilotriacetic acid (Ni-NTA) and nickel iminodiacetic acid (Ni-IDA). NTA and IDA fix transition metal ions to the matrix, where they complex polyhistidine sequences on proteins of interest. IDA is trivalent, meaning it possesses three metal binding sites, whereas NTA is tetravalent. Choice of resin modification affects product yield and quality. With four binding sites, NTA binds more strongly to polyhistidine but results in lower yields. IDA binds less strongly. Accordingly, it is less specific, but protein yields are higher.

A company specializing in IMAC resins for purifying polyHIS-tagged proteins is Cube Biotech. The company’s NTA and IDA resins work not only with nickel, but also with cobalt, copper, zinc, iron, and aluminum. Each of these cations brings unique polyhistidine-binding capabilities.

“There’s always a compromise between specificity and yield,” explains Ute Boronowsky, Ph.D., head of sales and marketing at Cube Biotech. Copper shows low specificity and high yield, whereas cobalt is on the other end of the spectrum, generating extremely pure proteins but lower yield. “Nickel is viewed as the best compromise,” notes Dr. Boronowsky. “Cobalt is second in terms of popularity.”

Where yield and purity are issues, particularly in very small or dilute samples, Dr. Boronowsky recommends forgoing resins in favor of magnetic beads, which provide easier separation of the final product.

IMAC resins are also used in proteomics. Zinc-binding proteins such as zinc finger proteins can be captured by zinc-bound resins. Phosphorylated proteins can be captured by iron, gallium, and aluminum resins. “It is also possible to capture the entire population of proteins in cells that bind to metals such as copper or iron, the so-called metalloproteome,” adds Dr. Boronowsky.

IMAC resins require only two histidines for binding. Six to eight histidines are typically used to increase specificity over intrinsic histidine in contaminating proteins. However, according to Dr. Boronowsky, when membrane proteins are being investigated, 10 or even 14 residues might be employed to overcome the masking effects of any detergent that might have been added to improve protein solubilization.

Cleavage of polyhistidine from the protein is normally not attempted unless the protein is used as a drug in humans or animals. Regulators are concerned that the resident polyhistidine moieties could potentially bind nickel that has leached off a column. Additionally, polyhistidine might prove immunogenic or exhibit other undesirable pharmacology in vivo despite its apparent poor immunogenicity.

Recently, novel ligands have been developed that show significantly lower nickel leaching and better compatibility with EDTA and other chelating substances. This makes them suitable for the purification of proteins from eukaryotic samples, thereby extending the use of polyhistidine proteins from bacterial to more complex expression systems. However, the trade-off with currently available matrices (GE Healthcare’s excel and Roche’s cOmplete) is a reduced binding capacity compared to NTA or IDA ligands.

Cube Biotech offers PureCube resins for the purification of His-tagged proteins. The resins feature the two most commonly used chelating ligands: iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA). NTA is a tetravalent ligand providing greater specificity but lower yield, while IDA is trivalent—less selective but generally providing more protein. Both IDA and NTA can be loaded with the metal ion most suitable for a given application.

Interesting Spin-Offs

Several interesting applications have spun off of traditional polyhistidine tagging. Maine Biotechnology Services, for example, offers MBS MAB230P, a polyhistidine-tag-specific antibody for confirming the presence of polyhistidine-tagged proteins in cell lysates through Western blot assays. “Many customers use it for research- and larger-scale assessment of recombinant protein expression,” says Cheryl Steenstra, Maine Biotechnology’s program manager for custom services.

ForteBio, a Pall business unit, has incorporated MAB230P into its second-generation Dip and Read™ Anti-HIS fiber optic biosensor. This biosensor, the tip of which is coated with the MBS antibody, directly captures and detects polyhistidine-tagged proteins for rapid quantitative measurement.

When binding to the polyhistidine-tagged proteins occurs, an interference pattern of light is reflected from the fiber’s surface, allowing monitoring of binding in real time using a proprietary ForteBio detection system. Protein concentrations may be read off a standard curve of known concentrations of polyhistidine-tagged proteins. ForteBio has sensors optimized for both hexa-histidine and penta-histidine tags.

Similarly, R&D Systems offers “His Tag Horseradish Peroxidase-conjugated Antibody,” which recognizes polyhistidine at either the N- or C-terminus of labeled proteins. Other vendors of antipolyhistidine antibodies include Cell Signaling Technology, Thermo Fisher Scientific, Abcam, Santa Cruz Biotech, Qiagen, and BioLegend. All such products are optimized for polyhistidine-tagged protein detection through Western blots.

Maine Biotechnology’s core business is antibody and immunoassay development. The company’s recombinant expression services are primarily focused on generating the immunogen and screening reagents to support the development efforts of their customers. The company maintains an inventory of polyhistidine-tagged protein controls as well. These are used in part to ascertain that antibodies the company develops for customers are specific for the target protein and not the polyhistidine sequence.

Maine Biotechnology Services, which offers bacterial recombinant protein expression as a service, asserts that in recombinant antigen projects related to hybridoma development, the applications customers have for their antibodies can be considered during every phase of antigen production. The company’s MAB230P monoclonal antibody to the polyhistidine tag is commonly utilized throughout the development process as an integral detection tool.

Several Maine Biotechnology customers also use the MAB230P antibody as a ligand on affinity resins. “But nickel column affinity chromatography is a well-established method, so our customers use the antibody mostly for detection” Steenstra notes. A benefit of polyhistidine-specific antibodies is that MAB230P needs to “see” only four histidine residues. “Quite often the tag, either at the C- or N-terminus, can be partially buried and not fully accessible,” Steenstra adds. “MAB230P can bind to a shorter, 4X HIS epitope.”

For its own antibody development programs, Maine Biotechnology sometimes constructs polyhistidine tags of up to 10 histidine residues for relatively insoluble proteins. Each recombinant protein target is subjected to solubility prediction models. “If it appears that it will be expressed in lower yield in the soluble fraction, extending the length of the polyhistidine fusion tag is one way to boost capture efficiency and purity of the isolated protein,” Steenstra observes.

Although polyhistidine tagging is the company’s workhorse fusion tagging method, Maine Biotechnology also employs maltose-binding protein and glutathione-S-transferase fusion proteins. The issue with these constructs, however, is that they tend to be large. With larger constructs, there is a greater likelihood that the antibodies to the proteins tagged with those sequences will react more to the tag than to the protein. Polyhistidine is small enough, and insufficiently immunogenic, to allay these concerns.


Large-Scale Ready?

Whether polyhistidine tagging is capable of production-worthy separations is a question that arises from time to time. According to Grigoriy Tchaga, Ph.D., R&D director at BioVision, “this is a long-stretch proposition, although some purification resin makers are preparing for this eventuality.” Resin manufacturer Qiagen, for example, already has documentation on file with the U.S. Food and Drug Administration, in the event that a drug developer attempts to use polyHIS as a purification modality.

Two major issues with large-scale purification are toxicity and immunogenicity. Nickel, the most popular immobilized metal for polyhistidine protein purification, is quite toxic, and it could leach from affinity columns. Cobalt is slightly less toxic. Zinc, biologically speaking, would be best. Zinc ligands exhibit provide specificity and yield.

The hexahistidine tag itself, in addition to potential immunogenicity problems, can change the nature of proteins. “In some cases, introducing excessive positive charge on the C- or N-terminus causes solubility problems and aggregation,” says Dr. Tchaga. “Also, it may increase the potential for immunogenicity.”

According to BioVision, prospects for production-scale polyhistidine purification depends on reducing toxicity and immunogenicity, much of which is due to leaching of immobilized metals from affinity columns. BioVision suggests that zinc would be a less toxic metal than nickel or cobalt. In this image, a tetradentate ligand-immobilized Zn2+ ion is shown binding a polyhistidine-tagged protein.

The solution may be to duplicate the histidine patterns on naturally occurring polyhistidine peptides, which are rare. Dr. Tchaga worked on these when he was R&D director at Clontech Laboratories. “There are some proteins,” he asserts, “that are better expressed and more soluble when they carry natural polyhistidine tagging compared with the artificial hexahis tag.”

Additionally, the energy requirements for an expressing organism like Escherichia coli to add six histidine residues during translation are higher than for those naturally occurring sequences.

Polyhistidine tagging may yet be taken seriously as a large-scale purification modality, provided biopharmaceutical companies see at least two key developments: 1) resins that provide higher selectivity than that available with current nickel and cobalt resins; and 2) purification tags that elicit little or no immune response. “We need this combination,” insists Dr. Tchaga, “for someone to invest significantly in large-scale polyhistidine purification for proteins with therapeutic applications.”

Losing the Charm

Polyhistidine tagging has become the leading technique for small-scale protein purification, but according to Ake Danielsson, Ph.D., R&D director at GE Healthcare, it is not a “one-size-fits-all method.” Although the tradeoff between protein purity and yield is recognized, the relationship between the two is subtle. Sometimes IMAC chromatography does not provide sufficient purity—regardless of the yield—for scientists to have absolute confidence in their protein product.

“Single-step purification may be warranted in many instances, but scientists now routinely add a second purification step, typically based on size-exclusion chromatography, to achieve their target purity” says Dr. Danielsson. The two-step approach is called for when protein impurities might interfere with the activity of the target protein.

During the past year, GE Healthcare has worked on developing prepacked size-exclusion chromatography columns for purification of recombinant proteins, including polyhistidine-tagged molecules. The columns have a diameter of 10 mm and a length of 300 mm. The product launch is expected in Spring 2016.

Dr. Danielsson admits that adding a second step diminishes the charm of polyhistidine tagging and one-step purification, but calls the two-step approach a compromise with reality. “Size-exclusion chromatography is a generic, fool-proof method,” he comments. “It’s sensible to do when impurities are a potential issue.”

New researchers in particular may become disillusioned with IMAC if they expect more from the purification medium than can be reasonably expected. “You must realize that IMAC is a good starting point,” advises Dr. Danielsson. “But by no means does it represent an optimized purification protocol.”

Dr. Danielsson also sees a trend, both in academic institutions and companies, to delegate protein purification to dedicated, core groups. He mentions universities in Singapore and in Sweden that have already adopted this model, as well as corporations that have embraced it for operational efficiency. Fueling this tendency is the standardization of purification regimens as platforms, similar to the current philosophy with regard to large-scale monoclonal antibody manufacturing. IMAC chromatography is firmly established in that regard.