A protein tag is an amino acid sequence that is attached to the N- or C-terminus of a recombinant protein through genetic engineering. It is most commonly exploited to facilitate protein purification or detection, yet protein tags can also be used to localize a protein to a particular cellular compartment, to improve solubility, or to enhance protein stability by preventing proteolysis. The selection of an appropriate protein tag is largely dependent on the nature of the target protein itself, and should take into account features such as the stability and hydrophobicity of the protein, in addition to its molecular weight. The intended downstream application of the tagged protein should also be given due consideration.
Researchers are now turning to new and innovative protein tagging methodologies.
An extensive range of protein tags exists, with Green Fluorescent Protein (GFP), Glutathione S-Transferase (GST), FLAG, hemagglutinin antigen (HA), poly-His, c-Myc, Maltose Binding Protein (MBP), and V5 being among the most well-known. All of these have been extensively literature cited, yet each tagging method has its own associated advantages and disadvantages; while many of the tried and tested protein tags remain popular, researchers are now turning to new and innovative protein tagging methodologies.
The HiBiT protein tagging system from Promega. Endogenous protein expression can be easily measured by combining HiBiT tagging with CRISPR/CAS9 gene editing.
One company pioneering these tags is Promega, whose HiBiT protein tagging system (launched in August) recently won a MipTec product innovation award in recognition of achievement in the development of novel technologies. The HiBiT tag is composed of just 11 amino acids, giving it a molecular weight of 1.3kDa, and is easily added to a target protein through traditional methods or CRISPR/Cas9 gene editing; when combined with the appropriate detection reagent, the resulting bioluminescence is sensitive enough to measure endogenous protein expression in minutes without the need for antibodies.
According to Amy Landreman, global product manager at Promega, HiBiT is an optimal tag for simple, rapid quantification of cellular proteins since its extremely small size means that it has negligible effect on cellular processes. “Once the HiBiT tag has been added to a protein of interest,” says Landreman, “it can be easily quantified using a detection reagent containing its complementary polypeptide and substrate to produce extremely bright luminescence. This is directly proportional to the amount of HiBiT-tagged protein expressed in the cell over seven orders of magnitude, and with a signal stability of several hours the HiBiT protein tagging system is scalable to high-throughput applications.” The HiBiT system also has utility for Western blotting, providing femtogram sensitivity without the need for laborious multi-step antibody-based staining protocols, and can be used in a live-cell format for specific detection of cell surface protein expression.
Another protein tag that does not interfere with structural or functional studies due to its small size is Strep-tag™ II*, an eight amino acid tag that has high affinity for a specialized StrepTactin™* ligand. Emma Lind, product manager at GE Healthcare Life Sciences, says that in the majority of cases it is not necessary to remove the tag from the protein of interest before performing a study, and since the binding affinity of Strep-tag™ II to the StrepTactin ligand is so high (almost 100-fold greater than the affinity for streptavidin), the system provides a highly efficient method of protein purification. “StrepTactin™ bound to Sepharose High Performance chromatography matrix gives high-resolution separations, sharp peaks, and purified target proteins in a concentrated form,” explains Lind.
“StrepTactin Sepharose High Performance chromatography resin is compatible with a wide range of additives, tolerates all commonly used aqueous buffers, and is quickly and easily regenerated using 0.5M NaOH. Purification of Strep-tag II-labeled proteins is performed under physiological conditions, while the elution process is very mild, simply relying on the use of desthiobiotin to displace bound proteins at the biotin-binding site of StrepTactin; these features allow the activity of the target protein to be preserved.”
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Although many single step protein purification processes are highly efficient, multi-tagging of proteins is an increasingly popular method for further enhancing purity. By expressing two different protein tags on the recombinant protein, it is possible to perform consecutive purification steps, resulting in an ultrapure protein preparation. This method is known as tandem affinity purification (TAP), and was first described almost 20 years ago when Rigaut et al purified a protein that they had tagged with both Protein A and a calmodulin binding peptide. Proteins purified in this manner are ideally suited to the study of protein-protein interactions, since the method minimizes the likelihood of any contaminating protein being present in the sample.
The InterPlay Mammalian TAP System from Agilent Technologies allows quick and easy recovery of interacting proteins from mammalian cells. The protein of interest is fused to a streptavidin binding peptide and a calmodulin binding peptide, and is subsequently purified by TAP. According to Laura Whitman, global product manager at Agilent, “our kits contain the necessary vectors, resins and buffers for mammalian TAP purification, resulting in exceptionally pure protein for downstream applications. We also offer an InterPlay adenoviral TAP system that combines our TAP system with our exclusive adenoviral gene delivery system, the AdEasy Adenoviral Vector System, for enhanced gene delivery to a broader range of mammalian cells, higher protein yields, and improved purification and analysis of endogenous interacting protein partners.”
Clive Mason, director of biology at Discuva, has extensive experience with protein purification techniques. “For an affinity tag system to have the best utility,” notes Mason, “the tag should exert minimal effect on the structure and biological activity of the expressed protein. Furthermore, it should afford a simple (ideally one-step) purification process, and should be easily cleavable, allowing the native protein to be recovered. A good tag also permits the recombinant protein to be assayed to enable tracking during the purification process.”
Of course, once any tagged protein has been purified, it is necessary to confirm that the purification process has been successful. This can easily be determined by, for example, Western blotting, dot blotting, or ELISA, and a highly cost-effective control for these assay formats is Absolute Antibody’s Multi-Tag control protein. This product, says Ian Wilkinson, head of research and manufacturing at Absolute Antibody, “is composed of seventeen commonly used protein tags fused together using flexible linkers. Although it was originally developed as an internal QC tool for our comprehensive panel of anti-tag antibodies, we’ve experienced high demand from our customers, and we believe it to have more tags in it than any other protein available elsewhere”.
With such an extensive range of protein tags to choose from, there is a suitable option for almost every scenario. While many protein tagging methodologies have been widely endorsed through publication, many more techniques are becoming mainstream, and with new protein tags continually being developed their utility continues to grow.
*These trademarks are owned by IBA GmbH.
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