Protein Purification: Tag You’re It!

Abstract

Molecular tags for purifying proteins have been around since the 1980s, when protein A was used to tag target proteins, which were purified with affinity chromatography using IgG resin. But by today’s standards, protein A is a large, unwieldy tag at 280 amino acids. Although large molecular tags can lend greater solubility to target proteins, the steric hindrance they can introduce may interfere with normal protein function. Here’s a look at some of the more commonly used protein purification affinity tags utilized by researchers.

His tag

The small polyhistidine tag (usually six histidine residues) has minimal effects on protein function and is widely used for recombinant protein production in all common expression systems. “An increasingly common sample type is cell-culture fluid containing secreted tagged target proteins from insect cells,” says Åke Danielsson, research director at GE Healthcare Life Sciences.

Proteins with His tags can be purified using immobilized metal affinity chromatography (IMAC). The histidine residues bind to transition metal ions immobilized on the resin; nickel and cobalt are most commonly used. “Although His-tagged proteins bind with a slightly lower efficiency to cobalt-chelating resins than to nickel-chelating resins, there is also a significant reduction in nonspecific binding [to cobalt resins],” says Colin Heath, R&D and marketing director at G-Biosciences. Cobalt resins have a higher selectivity for His-tagged proteins, but they also have a lower loading capacity, says Heath, and “therefore cobalt resins should be used for valuable recombinant proteins in limited quantities.”

IMAC resins for purifying His-tagged target proteins are widely available, including from Clontech, G-Biosciences, GE Healthcare Life Sciences, QIAGEN and Promega.

GST tag

Another popular tag is glutathione S-transferase (GST), a 26-kDa sequence. Although one of the larger tags, it has been demonstrated to make certain target proteins more soluble, while maintaining protein function. Solubilizing membrane proteins for purification can be particularly challenging, so a tag that helps can be welcome. “Sometimes the tag is hidden in the detergent shield that covers the membrane protein, so the protein will not bind to the resin and will not be purified,” says Danielsson.

GST-tagged proteins can be purified using glutathione resins. Tools for GST tags are available from many companies, including G-Biosciences, GE Healthcare, MilliporeSigma, Promega and QIAGEN.

MBP tag

Maltose-binding protein (MBP) is another large tag (45 kDa) that can interfere with protein function, but advantages include possibly increasing a target protein’s solubility and even its expression level. MBP-tagged proteins can be purified using amylose resins. Tools for MBP tags are available from several vendors, including GE Healthcare, GenScript and New England Biolabs.

CBP tag

The calmodulin-binding peptide/protein (CBP) tag (26 amino acids; 4 kDa) consists of a portion of muscle myosin light-chain kinase. At low calcium levels, it binds to calmodulin; upon removal of calcium, a conformational change in calmodulin forces the release of the CBP tag. The small size and mild elution conditions of the CBP tag make it amenable to studying the functions of purified proteins. Tools for CBP-tagged proteins are offered by several companies, including Biovision, G-Biosciences and GenScript.

Streptavidin/biotin-based tags

The natural, high affinity between biotin and avidin/streptavidin makes this protein pair extremely valuable for tagging proteins.

Target proteins tagged with a BCCP sequence of amino acids can then be biotinylated on that site by the enzyme biotin ligase. After tagging with biotin, the target protein can be purified using an avidin resin. Variations of avidin/streptavidin resins have been created that offer different affinities for the biotin tag, which can be useful, depending on the target protein’s properties.

One example is Strep-tagII, an 8-amino acid peptide that binds to Strep-Tactin in the same place that biotin binds streptavidin. The Strep-tagged proteins are isolated using resins containing Strep-Tactin or Strep-Tactin XT, which are engineered forms of streptavidin. Target proteins are then eluted using desthiobiotin or biotin, respectively, which have a higher affinity for the immobilized phase than does Strep-tagII. “The mild, physiological conditions used in purification and elution of Strep-tagII fusion proteins make the proteins suitable for structural and functional investigations, protein-protein interactions or ligand-receptor investigations,” says Ricarda Busse, product manager for protein production and assays at IBA Lifesciences.

IBA Lifesciences offers a newer Twin-Strep-tag, which consists of two Strep-tagII tags in series connected by a GS-linker. “The near-covalent affinity of Twin-Strep-tag to Strep-Tactin XT can be used to efficiently immobilize proteins for assay development,” says Busse. The lab of Alexei Yeliseev, staff scientist at the National Institute of Alcohol Abuse and Alcoholism at the National Institutes of Health, uses IBA Lifesciences’ Strep tag tools for purifying integral membrane G-protein-coupled receptors, such as the cannabinoid receptor CB2. The low expression level and hydrophobicity of CB2 presents solubilization and purification challenges that Yeliseev’s group is tackling using IBA’s Twin-Strep-tag. “We found that a double repeat of the Strep-tag is much more efficient for capturing CB2,” he says, adding that a third Strep-tag did not further improve CB2 recovery.

Yeliseev’s group also benefits from IBA’s new Strep-Tactin XT resin, which has a reduced off-rate of the bound tag. “This means that the resin can be used directly for capture of the tagged protein from very dilute solutions,” says Yeliseev. “We use it in our most challenging purification projects.” He also likes the fact that tags can be placed at either end (or even in the middle) of target proteins, that tags do not interfere with CB2 expression levels, and that mild elution conditions preserve the purified CB2’s receptor activity.

Tools for Strep and biotin tags are available from many vendors, including Bio-Rad Laboratories, G-Biosciences, GE Healthcare Life Sciences, IBA Lifesciences, MilliporeSigma and QIAGEN.

Epitope tags

Epitope tags are typically short sequences added to a protein that act at epitopes, or antibody recognition sites. The epitope-tagged protein can be purified by affinity chromatography with immobilized primary antibodies. MilliporeSigma’s FLAG® Expression System labels proteins with an 8-amino acid FLAG tag and uses monoclonal and polyclonal antibodies specific for the FLAG sequence for purification. “We provide the antibody pre-attached to a variety of different immobile phases, such as beads or resins, for convenient separation,” says Isaku Tenida, head of protein and pathway technologies at MilliporeSigma. The FLAG system is suitable for many types of proteins, but Tenida notes that complications can arise for proteins “that are highly insoluble or have a tendency to aggregate during expression.”

Other common epitope tags include hemagglutinin HA, c-myc and T7. Using a primary antibody that’s specific to larger protein tags, such as His, GST or MBP, can also yield effective purification.

HaloTag

Promega’s HaloTag® system uses a 34-kDa tag that covalently binds to synthetic HaloTag ligands. “The covalent binding of HaloTag fusion proteins enables efficient protein recovery and allows stringent washing, which eliminates most nonspecific binding,” says Gary Kobs, senior global product manager at Promega. HaloTagged proteins are first captured using a HaloLink™ Resin. After washing, the target protein is released from the resin by application of a protease, which cleaves it from the HaloTag. “The covalent binding coupled with the low, nonspecific binding of the HaloLink Resin provides superior purity and recovery of recombinant proteins from cultured mammalian cells, even at low expression levels,” says Kobs.

The wealth of tools available for purifying proteins today makes it likely that you’ll find an affinity tag to suit your protein of interest. Given the range of solubilities that proteins exhibit, it’s nice to know that scientists have a range of tools to choose from.