In as an affinity tag for protein purification.

In 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). GFP is encoded by a single gene, gfp (717bp), which is cloned and inserted into a DNA sequence of interest (Tsien, 1998). The GFP is composed of 238 amino acids. GFP does not interfere with the cellular processes of the cell it is inserted into and it does not require a co-factor, besides oxygen (Jung et al., 2005). GFP is versatile and can bind to either the N or C terminus of the protein of interest (Wang and Chong, 2003). The behavior of the proteins upstream and downstream to the GFP determine if GFP should bind to the C or N terminus (Wang and Chong, 2003). The fluorescence of GFP is caused by a chromophore, which is located at residues 65-67 and is composed of Ser65, Tyr66, Gly67 (Jung et al., 2005). The chromophore is the part of the protein that is responsible for absorbing and emitting light when excited and requires molecular oxygen to function, limiting the use of GFP to aerobic conditions (Tsien, 1998). The wild type (wt) GFP has a green fluorescence emission that peaks at 510 nm when excited by a wavelength of 400nm (UV light) and is the colour green (Jung et al., 2005). In genetically engineered versions of GFP, there are several different mutations that affect the chromophore’s behavior causing GFP to emit different colours and a higher brightness. Mutations of the Ser 65 form an Enhanced Yellow Fluorescent Protein (YFP) (Jung et al. 2005). Different coloured GFPs such as YFPs are beneficial when tracking the interaction of 2 or more different proteins. Another mutant of GFP is GFPuv, which is able to absorb a lower UV light wavelength of 395nm which contains more energy than a 400nm wavelength and produces 18 times the fluorescence as wt GFP (Penna et al., 2004). GFP has many uses; to track movement of proteins in cells, to view binding sites of proteins and to determine gene expression when GFP is controlled by a promoter.GFP protein tagging is one of the most used methods of examining live protein movement such as Amyloid Precursor-like Protein (APA1) in Caenorhabditis elegans (Wiese et al., 2010). Researching Alzheimer’s diseases (AD) is difficult as it only exists in humans, is costly and sometimes inhumane (Hargins and Blalock, 2017). APA1 is of interest to Alzheimer’s research because it is very closely related to Amyloid Precursor Protein (APA), which in humans, when cleaved, causes AD (Wiese et al., 2010). GFP proteins were used to track APA1 in C. elegans to determine their behavior and localization in the species (Wiese et al., 2010). It was discovered that GFP tagged APA1 were found in the cells of the worm that contained synapses and the removal of APA1, similar to cleaved APA, caused a deficiency of the synaptic function (Wiese et al., 2010). The researchers concluded that APA1 shows promise in the research of AD (Wiese et al., 2010).