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
Angelo DePalma, Ph.D.
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).
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.
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.
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.