Last updated: September 9, 2019
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Protein tagging is of great importance in molecular biology research and experiments (Chase and Kuo, 2010). In research, two protein tags, specifically Histidine (His-tag) and Green Fluorescent Protein (GFP), are used to study the function, cellular pathways, and interactions between proteins (Murayama and Kobayashi 2014). As protein tagging is employed more frequently, the knowledge on molecular and cellular biology concepts continue to expand.

His-tags are amino acid chains comprising of several successive histidine residues, most commonly six, incorporated into the N or C terminus of a target protein, dependent on how protein folds (Chase and Kuo, 2010). The His-tag is expressed in a vector, and fused in the frame of the protein of interest to facilitate protein purification (Chase and Kuo, 2010). Protein purification allows for the removal of weakly bound contaminants from the recombinant protein to study its structure and function (Ghahremanzadeh et al., 2017). Understanding the composition and physiology aids to determine therapeutic applications for a target protein (Ghahremanzadeh et al., 2017). Purifying a protein can be done via immobilized metal affinity chromatography (IMAC) (Chase and Kuo, 2010). IMAC is a technique that exploits histidine residues by separating them from His-tagged proteins (Ghahremanzadeh et al., 2017). Histidine consists of an imidazole side chain with electron donor groups that form coordination bonds with transition metals (Falke and Bornhorst, 2000). This forms the basis for His-tags high affinity for metal ions such as Cu2+, Co2+, Zn2+, and Ni2+ (Barbosa et al., 2015). On the charged resin of the IMAC column, immobilized Ni2+ ions form the strongest interaction with the histidine residues of the His-tag (Ghahremanzadeh et al., 2017). Ni(II)-nitrilotriacetic acid (Ni-NTA) is a metal-chelating agent that aids to chelate the histidine residues to nickel ions of the IMAC resin (Loughran and Walls, 2014). High concentrations of imidazole are added to the column to prevent non-specific protein binding to the IMAC resin (Chase and Kuo, 2010). This allows the six histidine residues to separate from the recombinant protein and remain bound to the Ni-NTA chelate group (Loughran and Walls, 2014). As the result, a purified protein can be eluted from the column (Loughran and Walls, 2014). Low pH can also be used to elute a purified protein from the column, however, it may damage the protein (Falke and Bornhorst, 2000).

The primary structure of GFP is a long polypeptide chain of 238 amino acids (Remington, 2011). The gfp gene consists of 714 base pairs that fuse with a protein of interest at its N- or C- termini, dependent on the functional domain of a protein (Murayama and Kobayashi 2014). When arranged into the tertiary structure, GFP forms an eleven stranded -barrel with an -helix inside (Remington, 2011). Three amino acids: tyrosine-66, serine-65, and glycine-67 are centered within the -helix (Remington, 2011). In the presence of oxygen, the amino acids get oxidized and form a mature GFP chromophore (Remington, 2011). For a wild type GFP, a chromophore can fluoresce green in its neutral protonated/A form or its anionic/B form (Remington, 2011). The A form absorbs ultraviolet light (UV) at approximately 395 nm whereas the B form absorbs light at approximately 475 nm (Remington, 2011). A genetically engineered GFP by mutagenesis, can give rise to different fluorescent variants such as blue, cyan, yellow, and red (Remington, 2011). A blue fluorescence occurs when Tyrosine-66 is replaced with the amino acid Histidine in GFP (Heim et al., 1994). Tracking a GFP has advanced studies in vivo protein visualization and localization, gene expression, and protein-protein interactions (Heim et al., 1994).

In 2009, a pandemic outbreak known as swine flu (H1N1 and H3N2 influenza virus) infected a large proportion of the human population (Bestebroer et al., 2011). Therefore, the need for an influenza vaccine was at an all-time high to cease the ever-growing human cases (Bestebroer et al., 2011). Recombinant influenza viruses were constructed to carry the swine subtype, H1N1 (Bestebroer et al., 2011). Upon infection, reverse genetic plasmids were used to clone the GFP gene in the neuraminidase (NA) gene at the 3′-5′ termini (Bestebroer et al., 2011). Various animal species were experimentally infected with the GFP-expressing viruses and a fluorescence imager was used to track the localization within the specimen (Bestebroer et al., 2011). Titers were obtained from the specimen infected with the GFP-expressed virus strains (Bestebroer et al., 2011). It was discovered that antibodies directed to the H1N1 and H3N2 influenza viruses (Bestebroer et al., 2011). Thus, as a result of the expression of GFP in influenza viruses, influenza vaccines were brought to clinical trials (Bestebroer et al., 2011).