Purifying biological macromolecules like proteins is a mainstay of biotechnology and pharmaceutical industries. Affinity tags are an efficient strategy for purifying recombinant proteins, allowing the tagged portion of the recombinant protein to be efficiently purified using an affinity matrix. This blog reviews the most commonly used affinity tags and details newer affinity tag technologies.
What is an affinity tag?
Affinity tags are enzymes, protein domains or small polypeptides that bind substrates with high specificity to enable the rapid and efficient purification of proteins. Substrates include carbohydrates, small biomolecules, metal chelates, and antibodies. Affinity tags can be used for both prokaryotic and eukaryotic protein expression systems.
The first affinity tag for the purification of recombinant fusion proteins was described in 1985. Since, affinity tags have been widely used, with a search for “affinity tag” in Google Scholar yielding more than 30,000 publications1. For the Human Protein Atlas (HPA), polyhistidine tags were used to generate more than 50,000 recombinant proteins1. The ultimate deciding factor for choosing affinity tags is ensuring rapid and efficient protein purification, which results in high expression levels and high purity of the target protein. In addition to allowing facile purification of a target protein, affinity tags can also improve solubility. The most commonly used affinity tags are summarized in the table below (Table 1).
Table 1. Widely used affinity tags
| Affinity tag | Size (kD) | Affinity matrix |
| Hexahistidine (6x His) | 0.84 | Metal ions (Ni2+, Co2+, Cu2+, Zn2+, Fe3+) |
| Glutathione S-transferase (GST) | 26 | Glutathione |
| FLAG | 1.01 | Anti-FLAG mAb |
| Streptavidin-binding peptide (SBP) | 4.3 | Streptavidin |
| Strep II | 1.06 | Strep-Tactin (modified streptavidin) |
| Maltose-binding protein (MBP) | 42 | Amylose |
| Calmodulin-binding peptide (CBP) | 2.96 | Calmodulin |
| Chitin-binding domain (CBD) | 5.59 | Chitin |
| S | 1.75 | S-protein of RNase A |
| HA | 1.1 | Anti-HA epitope mAb |
| c-Myc | 1.2 | Anti-Myc epitope mAb |
| HaloTag® | 33 | HaloTag® ligand (containing chloroalkane + functional group) |
Affinity tag purification workflow
The first step in an affinity tag protein purification workflow is the expression of a recombinant protein with an affinity tag. Affinity tags are cloned into either the N- or C-terminus of a target protein by including the cDNA sequences encoding the tag peptide into a matching reading frame of the target protein. Depending on the size and potential immunogenicity of the affinity tag, the tag may need to be removed after the initial purification of the tagged protein. In this case, a spacer (only for N-terminal tagged proteins) contains an endopeptidase cleavage sequence.
The purification process can begin once the tagged protein is expressed in the expression system, and the appropriate cellular components are obtained. For this, affinity chromatography is employed, which relies on the specific binding of the affinity tag to a ligand bound on an affinity matrix — made of an affinity resin. Typical examples are described below. Proteins not containing the affinity tag have a weak affinity for the affinity matrix, and non-specific binding contaminants can be removed using wash steps appropriate for the protocol and application. An elution step allows the chemical bonds binding the tagged proteins to the affinity matrix to be broken, releasing recombinant proteins and interacting protein partners. Additional steps include the removal of the tag with endopeptidases.
Applications for affinity tag purified proteins
Following affinity tag purification, a series of methods (SDS-PAGE, western blots) are used to validate the purity and yield of the target protein. Protein complexes can be identified following pulldown assays of the target protein and associated protein binding partners, which often use mass-spectrometry techniques. Structural analysis can also be performed on highly purified proteins to study primary, secondary and tertiary protein structures, including NMR and crystallography. Protein-protein interactions and determination of kinetic and thermodynamic parameters can be studied using surface plasmon resonance (SPR) immobilization strategies. Another critical application of affinity tags is cellular imaging.
Examples of affinity tags
This blog reviews the most commonly available tags (His and GST) regularly used in several applications in standard laboratory settings. We also include examples of more recently developed affinity tags (HaloTag, Chromotek).
His-tag
The His-tag takes advantage of the natural ability of the histidine amino acid to coordinate strongly with metal ions. Developed in 19883, polyhistidine tends to consist of six consecutive histidine residues, but His-tags can vary between two to ten histidine residues. His-tags are chosen for their small size and high efficiency in purifying proteins. The affinity matrix relies on the interaction between the imidazole ring of histidine on the polyhistidine fusion proteins and metal ions – immobilized divalent metal ions (Ni2+, Co2+, Cu2+, Zn2+, Fe3+), with Ni2+ predominately used. Interaction between the His-tag and metal affinity matrix occurs at neutral to slightly basic pH (7.5-8), with the binding affinity affected by the length of the His-tag (higher binding affinity with longer His-tag compared to shorter ones). The His-tagged protein can be eluted from the affinity matrix by decreasing the pH to 4-5. Competition with imidazole is also used to elute the His-tagged protein, which is also included at lower concentrations in the binding and wash buffer solutions to avoid non-specific binding with histidine-containing proteins. The inclusion of stringent wash conditions with imidazole, which has the same structure as histidine, is essential when purifying His-tagged proteins from insect and mammalian cells, which contain a higher percentage of histidine residues in their proteins than E. coli cells and can cause significant background binding4. The His-tag is a cleavable tag that can be removed using site-specific proteases. The conditions for each His-tagged experiment are protein-dependent; each experimental step must be determined empirically, and reactions must be scaled up linearly for larger or smaller quantities and reaction volumes.
GST-tag
The GST-tag exploits the high affinity of Glutathione S-transferase (GST) towards reduced Glutathione. GST is a naturally occurring 26kD enzyme involved in detoxification by catalyzing the conjugation of the reduced form of glutathione to xenobiotic substrates. Expression of recombinant proteins with a GST-tag is often favored because of the high-affinity binding of GST to glutathione coupled to a Sepharose matrix. Elution of the GST-tagged proteins is enabled using a mild, non-denaturing buffer containing reduced glutathione. Like the His-tag, the GST-tag can also be removed. The advantage of employing the GST-tag is the high expression levels achieved in E. coli cells (typical yields ~ 10mg/litre), resulting from aggregated protein accumulation in inclusion bodies. This effect leads to the drawbacks of the GST-tag, which involves refolding insoluble fusion proteins and buffer exchange before purification. Furthermore, some insoluble proteins may not refold correctly4. Additionally, the GST-tag tends to dimerize in solution, which may affect the properties of the target protein4.
HaloTag
Although efficient for the isolation and purification of proteins, the drawback of affinity tags like His-tags is that additional tagging systems are needed for cellular imaging applications. To overcome these limitations and allow researchers to use one single construct to perform multiple applications, the HaloTag system was developed in 20085. The HaloTag is based on the formation of a highly specific and irreversible covalent bond. The 33kD HaloTag is fused to the target protein. The molecular mechanism underlying the HaloTag is based on a mutation in the bacterial haloalkane dehalogenase enzyme from Rhodococcus rhodochrous, where Phe272 is replaced with His272. The substitution of this residue allows irreversible binding with a HaloTag ligand and the formation of a covalent adduct with high stability5. The covalent bond between the HaloTag and HaloTag ligand is relatively stable under harsh conditions. Additionally, the HaloTag can be removed from the target protein as the HaloTag construct is readily digested by TEV protease treatment. Furthermore, additional affinity chromatography can remove the TEV protease from the purified target protein. Another advantage of the HaloTag is that a single construct can be used for different applications, including protein isolation and purification, evaluation of protein function, analysis of molecular interactions, protein assays, in vitro cellular imaging, and in vivo molecular imaging6.
Capturing tagged proteins using Chromotek’s NanoTraps for immunoprecipitation
Immunoprecipitation is another essential method of purifying tagged proteins, which employs antibodies that bind to the fusion tags. The problem with employing conventional antibodies is the target protein co-elutes with the heavy and light chains of the antibody used for purification. This high concentration of heavy and light antibody chains can contaminate the sample and obscure the identification of proteins in lower quantities, which represent physiologically significant interactions (in SDS-PAGE and mass spectrometry analysis and western blot detection). ChromoTek has developed Nano-traps to efficiently pulldown target proteins with fusion tags, like the GFP-tag, which are conjugated to beads so have no contamination of the Nanotrap. Alpaca-derived nanobodies only contain heavy chains and lack light chains. With a high affinity (dissociation constant kD 1 pM), ChromoTek’s Nano-Trap can efficiently purify GFP-fusion proteins, even when produced at low levels. Endogenously significant protein complexes can also be identified by employing Nano-traps in co-immunoprecipitation workflows.
LubioScience provides a comprehensive range of affinity tag reagents for protein purification applications. Please contact us for further details.
References
1. Human Protein Atlas. The Human Protein Atlas: A 20-year journey into the body. www.proteinatlas.org/download/HPA_-_a_20-year_journey_into_the_body.pdfhttps://www.proteinatlas.org/download/HPA_-_a_20-year_journey_into_the_body.pdf (2023).
2. Zhao, X., Li, G. & Liang, S. Several affinity tags commonly used in chromatographic purification. Journal of Analytical Methods in Chemistry vol. 2013 Preprint at doi.org/10.1155/2013/581093 (2013).
3. Hochuli, E., Bannwarth, W., Dobeli, H., Gentzi, R. & Stuber, D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology6, (1988).
4. Kimple, M. E., Brill, A. L. & Pasker, R. L. Overview of affinity tags for protein purification. Curr Protoc Protein Sci (2013) doi:10.1002/0471140864.ps0909s73.
5. Los, G. V. et al. HaloTag: A novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol3, (2008).
6. England, C. G., Luo, H. & Cai, W. HaloTag Technology: A Versatile Platform for Biomedical Applications. Bioconjug Chem26, (2015).
7. https://cube-biotech.com/knowledge/protein-purification/his-tag/
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