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 a
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).

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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).

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