March 1, 2015 (Vol. 35, No. 5)

Getting a Sure Hold on Protein Purification by Attaching Convenient Handles

Labile, poorly expressed, or poorly soluble proteins paose many difficulties in isolation and purification. These difficulties can be overcome, however, by various means. One approach is to modify recombinant proteins by means of fusion tagging, the addition of amino acids, functional domains, or whole proteins.

With fusion tagging, which has become a standard method in proteomics, the appended amino acid sequences, or tags, can act as purification “hooks” through their affinity to antibodies or metals. Rho1D4, for example, uses a hydrophilic sequence from the C-terminus of bovine rhodopsin followed by capture on affinity resins containing immobilized antirhodopsin antibodies.

Alternative affinity purification techniques rely on antibody-based capture. These techniques operate through a mechanism that is the opposite of that used to purify monoclonal antibodies by means of Protein A capture. That is, when proteins are to be purified, the antibody is the ligand and the protein is the target. Antibody-based protein purification methods are usually limited to isolating small quantities of purified protein.

By contrast, purification resins for proteins labeled with repeat polypeptide tags such as polyhistidine employ immobilized metal affinity chromatography (IMAC) through which coordination sites on Cu2+, Co2+, Ni2+, and Zn2+ attract electron-rich histidine residues. Proteins are “competed off” through an imidazole gradient or pH changes.

IMAC resin residues are far less expensive than antibody-modified resins and provide more regeneration cycles. But similar to other polypeptide tagging methods, polyhistidine tagging requires enzymatic removal of the tag, which adds a step to the process.

Purification of histidine-tagged proteins can be performed using GE Healthcare Life Sciences’ HisTrap™ columns and ÄKTA™ chromatography systems.

Polyhistidine Tagging

Developed during the late 1980s, polyhistidine tagging (PHT) is a mature technology that greatly simplifies protein purification. “It is by far the most commonly used affinity tagging technology,” says Åke Danielsson, staff scientist at GE Healthcare. “It is used for virtually all protein research areas. It is by no means a niche technology. It is big.”

One of PHT’s strengths is its ability to work at either the N- or C-terminus of most proteins. For most proteins, the N-terminus is the most common destination. In membrane proteins, however, the N-terminus is involved in protein insertion into the membrane.

Over the years, the number of histidine residues involved in PHT has ranged from 6 to 10. Investigators have their preferences, but the most common number used today is six. The histidine residues, sometimes along with a spacer, are added to the genetic sequence before transfection.

Nickel has emerged as the most common chelated metal in IMAC residues, but some researchers prefer other metal ions for specific purifications. For example, cobalt is considered superior by many for purifying membrane proteins. In the end though, researchers tend to use reagents that they consider familiar or can readily access.

IMAC resin evolution has tended toward greater stability and durability, higher capacity, and ability to withstand high flow rates. “We see this trend with all chromatography resins,” Danielsson observes.

More stable metal ion binding has enabled protein capture directly from the cell culture medium. This was not possible several years ago because media components had a tendency to strip nickel from resins, thereby destroying their affinity to polyhistidine.

Deep blue nickel-chelated columns turned white as media flowed through. Which components were responsible? Danielsson does not say exactly. Cell culture media manufacturers, GE Healthcare included, tend not to disclose the detailed composition of their media.

“Amino acids could be part of the explanation, but other factors may also contribute,” Danielsson confides. “A new generation of IMAC resins has been introduced to solve that problem. This has greatly facilitated the purification of HTPs produced by eukaryotic cells.”

Still, there are difficulties in applying HTPs to large-scale biomanufacturing of therapeutic proteins. “There are no registered processes for producing therapeutics in this way,” Danielsson admits. The main issue is cleaving off the polyhistidine tag. Cleavage is incomplete, and it often leaves behind one or more nonnative residues. For now, he says, HTPs are stuck in the manufacture of “for research use only” proteins.

A Hybrid Approach

The amino acids left behind after cleavage represent a significant drawback of polyhistidine tagging. The lack of histidine proteases forces the insertion of one or two amino acids between the polyhistidine moiety and the target protein. Proteases cleave at the juncture between this short linkage point and the polyhistidine, removing all histidine residues but leaving the anchor residue(s) behind.

Approximately 12 kDa in size, small ubiquitin-like modifier (SUMO) proteins attach to and detach from other proteins in cells. These actions of SUMO proteins are the basis of SUMOylation, a form of post-translational modification. It is relevant in altering protein function in various processes including protein transport, transcriptional regulation, apoptosis, protein stability, stress response, and advancing the cell cycle.

SUMOylation products are available from several vendors including LifeSensors, Life Technologies, Lucigen, and the nonprofit AddGene. SUMOpro™, a SUMOylation product from LifeSensors, enhances protein expression and solubility in Escherichia coli while simultaneously facilitating rapid purification. The company’s SUMOstar™ product works for purification from eukaryotic cells.

SUMO bridges the gap between pure polyhistidine tagging and alternative affinity tagging methods. It uses the SUMO modifier and cleavage enzyme but contains a polyhistidine region to facilitate purification through IMAC.

“If you cleave off most affinity tags with TEV [Tobacco Etch Virus] protease or growth factor 10, extra amino acids are left behind,” explains James E. Strickler, Ph.D., vice president of research and development at LifeSensors. “When you remove SUMO with SUMO protease, nothing is left behind but the native protein. And polyhistidine tags don’t provide a benefit toward improved folding or solubility, whereas SUMO does.”

According to Dr. Strickler, the number of papers on SUMO tagging is increasing. “But lots of people just make their own TEV protease, which is cheaper than buying from us,” he adds. Dr. Strickler also notes the importance of familiarity in helping investigators embrace SUMO: “If their colleagues have had good luck with polyhistidine tagging and TEV, they’ll use it. If they get great results with SUMO, they’ll proceed with SUMO.”

Barriers to adopting affinity tagging for large-scale biomanufacturing of therapeutic proteins are numerous and high. “It should work fine for therapeutic proteins except there are always questions about things leaking off of columns,” Dr. Strickler explains. A fully optimized SUMO or polyhistidine process, he estimates, would approximately break even in terms of cost compared with a typical three-column purification procedure.

But as always, the principal hurdle is regulatory risk. “Process-development folks are extremely conservative,” Dr. Strickler emphasizes. “They don’t want to be the first to seek approval for a particular column or method. It’s easier to fall back on methods that have already been approved.”

Making a Clean Break

“His-tagging remains the most popular affinity tagging method,” says Jens Stracke, Ph.D., sales director at Cube Biotech. “More than half of all research-stage proteins are purified through his-tagging because it’s cheap, robust, and easy to use.”

Besides offering products for this method, Cube Biotech specializes in customized affinity chromatography resins and magnetic beads for affinity capture. The company can also produce affinity resins based on customer ligands, and it sells activated agarose resins for do-it-yourself projects.

His-tagging really falls short, according to Dr. Stracke, with membrane proteins. For purifications involving these proteins, Cube Biotech sells a purification system called rho1D4, based on the last nine amino acids of the intracellular C-terminus of bovine rhodopsin. The rho1D4 matrix consists of an anti-rho1D4antibody coupled to agarose beads, and the corresponding tag easily incorporates into the recombinant protein of interest. The dissociation constant for rho1D4 is 500 times more favorable to binding than the polyhistidine-metal interaction.

The rho1D4 antibody and epitope have very high affinity—removing just two residues prevents binding. As a result, nonspecific affinity with sequences similar to rho1D4 is highly unlikely.

Once bound, protein is eluted by adding an excess of rho1D4 peptide, which binds competitively to the matrix antibody. “This provides for gentler elution conditions than, for example, changing pH,” Dr. Stracke says. For more than 20 years, membrane protein researchers have used this antibody matrix, but Cube Biotech was the first to commercialize it.

Because of the higher costs, Dr. Stracke advises customers to reserve rho1D4 purification for membrane or other difficult-to-purify proteins: “His-tagging is less expensive, and well suited for soluble proteins.”

Last but not least, despite the popularity of affinity agarose resins, Dr. Stracke notes a tendency to use magnetic beads bearing a suitable affinity ligand: “With magnetic beads you usually need less starting material and have the advantage of small elution volumes, which lead to higher concentrations of your protein of interest.”

Another sound alternative to polyhistidine tagging is multifunctional HaloTag® technology from Promega, which uses a 34 kDa tag. Like polyhistidine, HaloTag adds to the N- or C-terminus of the gene for the protein of interest. After expression and cell lysis, the HaloTag-modified protein forms a covalent bond with a 12-carbon chloralkane unit bound to the purification medium. Proteolysis with a HaloTag TEV protease cleanly cleaves the HaloTag and chloralkane, which remain on the resin. Purification media are available in magnetic and nonmagnetic formats.

The covalent bond is the hallmark of HaloTag. “The key benefit is that the tag is completely removed, so it cannot affect any downstream application,” notes Gary Kobs, proteomics marketing manager.

Like SUMO, HaloTag improves protein expression, folding, and activity. Additionally, the chloralkane capture moiety may incorporate fluorescent labels that allow not only the detection of protein-protein interaction but also other imaging-based assays. Introduction of other novel functionalities is possible through with succinimidyl ester, amine, iodoacetamide, or thiol reactive groups.

“It’s a multifunction tag, not just for purification,” Kobs explains. In fact, HaloTag was originally developed for imaging experiments and later modified for protein purification. 

HaloTag® technology, from Promega, is based on the formation of a covalent bond between a protein fusion tag and synthetic chemical ligands. By interchanging different synthetic ligands, researchers can control the function and properties of the HaloTag fusion protein.

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