His-tags added to the column to prevent non-specific

His-tags or
polyhistidine tags are amino acid chains comprising of approximately six
histidine residues incorporated into the N or C terminus of a target protein (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 of 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 Nickle ions (Ni2+)
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).