n a competitive inhibitor for his-tags, is then

n the last few decades, the field of molecular and cellular biology has vastly expanded due to the introduction and revolution of technology and laboratory techniques. One development is the implementation of cellular tagging, specifically Green Fluorescent Protein Tagging (GFP) and Histidine tagging (His-taging). Researchers tag proteins to gain an in-depth understanding of cellular function and composition of proteins.Histidine tagging is the process of ligating a genetic sequence, CAC or CAU repeats, that code for several histidines in a row, at the N or C terminus of a protein, after a start codon or before a stop codon respectively (Terpe, 2003). The placement on the N or C terminus of the his-tag is protein specific (Terpe, 2003). His-tagging is used as an affinity tag for protein purification. Protein purification is the process of isolating specific protein(s) from a mixture of proteins (Arnau et al., 2006). Immobilized metal affinity chromatography (IMAC), contains transition metal ions such as Co2+, Ni2+, Cu2+, Zn2+ that are immobilized on a matrix (Terpe, 2003). Out of all the amino acids, histidine forms the strongest bonds with transition metal ions due to electron donor groups on the histidine ring (Terpe, 2003). The more histidine residues in the his-tag, the stronger the affinity for the transition metal will be (Terpe, 2003). A strong affinity will result in a more pure protein (Terpe, 2003). If there too many histidine residues, the his- tag may be large enough to effect protein folding (Majorek et al., 2014). Proteins that contain a his-tag bind to the immobilized transition ions and stay in the matrix and the untagged proteins flow freely through the matrix (Terpe, 2003). A buffer that causes either a change in pH or contains high levels of imidazole, a competitive inhibitor for his-tags, is then passed through the matrix to remove the purified proteins (Terpe, 2003). After proteins are purified, a his-tag may interfere with the projected purpose of the protein (Arnau et al., 2006). His-tags can be removed using a specific endoprotease, which cleaves off the his-tag (Arnau et al., 2006). To remove the endoprotease from the mixture of pure proteins, the mixture is then run through another affinity purification as endoproteases contain also contain a tag (Arnau et al., 2006).