Tag! Purifying Proteins with Affinity Chromatography

IMAC ATTACK

Leading the affinity tag market is the polyhistidine (His) tag. Developed three decades ago by Jerker Porath and colleagues,³ His-tagging relies on the strong affinity that histidine's imidazole side chain has for metal ions such as nickel, zinc, copper, and cobalt. Most commercially available systems employ tags containing six to 10 tandem histidines and use nickel-based resins to purify proteins in a technique called immobilized metal ion affinity chromatography (IMAC). His-tagged proteins adsorb to columns under neutral or alkaline conditions and are eluted by competitive addition of imidazole or by low pH conditions, which can denature the protein.

According to one marketing report, nearly half of 580 scientists surveyed primarily use His tagging in their purification protocols.⁴ Brian Johnson, market segment manager for Rockford, Ill.-based Pierce Biotechnology, attributes the technique's popularity to its low cost, simplicity, and speed. And the tag's small size means it's not likely to greatly perturb protein structure. But IMAC does have some drawbacks: The metal ion can leach from the column, lowering protein yield, and the nickel resin can bind untagged, yet histidine-rich, proteins.

To address these problems, several companies have developed new twists to the traditional IMAC method. BD Biosciences-Clontech of Palo Alto, Calif., markets a TALON affinity resin, which uses a chelating ligand that holds cobalt ions with four bonds to eliminate metalion leaching. Cobalt ions have more affinity for histidine tags than do nickel ions, and they offer higher specificity, according to the company

Jutta Drees of Qiagen, the Netherlands, argues against cobalt. "Cobalt shows significantly higher leaching than the nickel does. If the cobalt is washed away, you lose more bound protein, and it is possible that you can copurify untagged proteins," she says. Qiagen's Ni²⁺-nitrilotriacetic acid resin has four chelating sites for nickel and can achieve 98% purity, Drees says. Users who want to remove the tag can employ Qiagen's TAGzyme system, which uses an exoprotease to cleave the His tag after purification.

© 1999 John Wiley & Sons

Before Cuatrecasas and Wilchek's invention of affinity chromatography protein purification protocols relied on biochemical characteristics. Ion exchange chromatography, for instance, (left) separates proteins based on charge. In gel filtration chromatography (middle), proteins are resolved by size, using porous beads that selectively slow the migration of appropriately sized molecules. The principle of affinity chromatography is diagrammed at right. (from: Cell &Molecular Biology, by G. Karp, 3rd Ed., 2003)

Bio-Rad Laboratories of Hercules, Calif., has a new Profinity IMAC resin that uses the company's UNOsphere polymer beads coupled to iminodiacetic acid, an ion chelator. Profinity's large, open-pore structure and relatively low ligand density set it apart from its competitors, according to the company. The open pores enable molecules to bind quickly even under high flow rates, which is an advantage for industrial applications. Low ligand density eliminates native His-containing proteins, "so when we do bind, we bind only the recombinant His-tag proteins, the target proteins," says Tanis Correa, Bio-Rad chromatography product manager. The resin is available uncharged or charged with nickel.

While IMAC and other affinity tag systems are ideal for a fast, effective first step, most scientists caution that they often must be followed by a second chromatographic step to remove salts and other contaminants. "The removal of a protein that is bound to an IMAC column requires imidazole for elution, which becomes in fact a contaminant that you may want to remove. And therefore you would need to have an additional step," says Van de Velde.

GST, TAP, AND OTHERS

Glutathione-S-transferase (GST) is another popular purification tag. Proteins fused to GST can be isolated on glutathione-coupled beads. But, weighing in at 220 amino acids, GST tags are significantly bulkier than their polyhistidine counterparts. The large size can cause recombinant proteins to form insoluble inclusion bodies that preclude purification under native conditions. Nevertheless, Simpson estimates that GST is the second most commonly used fusion tag.

Some tagging systems rely on the strong affinity of biotin for streptavidin and avidin. Biotinylated proteins can be purified on streptavidinylated resins from a variety of vendors, including Promega of Madison, Wis., Qiagen, and Pierce. Several different affinity tags are based on antigen-antibody interactions, including St. Louis-based Sigma-Aldrich's proprietary FLAG tag and the influenza hemagglutinin and c-Myc peptide tags from Abcam of Cambridge, UK. Another strategy uses the 4-kDa calmodulin-binding peptide (CBP), which binds to a calmodulin resin in the presence of calcium and enables purification of protein under neutral pH conditions.

In 1999, researchers at the European Molecular Biology Laboratory in Heidelberg, Germany, developed a dual-tag selection procedure called tandem affinity purification (TAP). Proteins to be purified are fused at either the N- or C-terminus to two tags (protein A and CBP) separated by a tobacco etch virus (TEV) protease cleavage site.⁵ After expression, low-abundance proteins can be purified from a complex mixture first by affinity chromatography on an IgG matrix (which binds protein A). The protein is then removed from the column under native conditions using TEV protease and incubated with calmodulin-coated beads in the presence of calcium to remove the protease and other contaminants.

The main advantage of TAP-tagging is that it purifies proteins under native conditions, thus ensuring that the resulting proteins can be used for functional assays. Additionally, since the two affinity tags do not need to be present on the same protein, the method can be used to copurify interacting proteins that contain one or the other tag. Stratagene of La Jolla, Calif., sells InterPlay TAP kits and vectors for this purpose. Qiagen sells its own Two-Step Affinity Purification System, which features vectors for producing proteins containing both His and Strep tags to facilitate sequential purification.

Currently more than 20 companies sell products for affinity chromatography; a dozen are listed in the accompanying table. Not bad for a technique that even the Nobel laureate whose name is on the first paper to describe it initially doubted. Anfinsen, who won the Nobel Prize for chemistry in 1972, "was skeptical of it," says Cuatrecasas. "He said he would put his name on the paper but hold his breath until someone repeated the work." Anfinsen didn't need to hold his breath for long. "If you put affinity chromatography in PubMed," says Wilchek, "you have at least 40,000 titles. ... There's no lab that doesn't use affinity."