Ultimately, and the breast cancer cells that went untreated

Ultimately, both green florescent proteins tagging andpolyhistidine tagging are both useful to monitor and track specific geneactivity. Both have their pros and cons as methods of immobilizedmetal-affinity chromatography, but with further research there can be improvementsin both techniques to improve the tracking of genes and protein-proteininteractions over time.

In research done by Coumans et al, human breast cancer cells wereanalyzed using green fluorescent proteins to track proteome changes. Breastcancer cells were cultured and treated with antibiotics under restrictedconditions and were then tagged with GFP (Coumans et al, 2014). The proteins were extracted after grown to a set sizeand analyzed using two-dimensional electrophoresis and isobaric tags forrelative and absolute quantitation (iTRAQ) (Coumans et al, 2014).  Comparisonswere made on cell behaviour and protein-protein interactions between the cellsthat were treated with antibiotics and tagged with GFP and the breast cancercells that went untreated (Coumans et al,2014). It was found that the antibiotic improved the outcome of the cells,but many results came up inconclusive (Coumans et al, 2014). There was proof of analytical variability andevidence of molecular changes caused by the GFP expression within the cell (Coumanset al, 2014). Within a protein, GFP can be fused to either the N orC terminus or between two proteins to study the protein-protein interactions butthe insertion must be done strategically so there is a lack of disruption tothe natural reading frame (Catanzariti etal, 2004). Due to its fluorescence,scientists can isolate where the gene is expressed and when GFP is triggered byblue ultraviolet light and produces a green fluorescent light.

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When a lightwith a wavelength of 400nm strikes the chromophore, the protein goes into anexcited state (Heim et al, 1994). Whenthe chromophore enters ground state, the light emitted will be at thewavelength 505nm (Heim et al, 1994).This light appears on the visible light spectrum as a green colour (Heim et al, 1994).

The green fluorescent protein (GFP) is a bioluminescentprotein that was first discovered within Aequoreavictoria, which is a species of jellyfish (Prasher et al, 1992). The green fluorescent protein works be producing afluorescent glow in vivo allowing scientists to study the dynamics of theprotein under a microscopy without altering or killing the cell (Tsien, 1998). Withina plasmid, GFP can be placed subsequent to the promoter region and beside thegene of interest. The restriction enzymes can cut the section of interest outand then add the enzyme ligase and then the plasmid can be added to a protein (Tsien,1998).  Histidine tagging is a method of IMAC that is verysimple to use and can achieve a significant increase of purification in asingle step (Kaake et al2010).

The polyhistidine residue mustfirst be fused to the gene of interest within a plasmid (Singh and Jain, 2013).The plasmid must be capable of replication, contain a gene available for theallowance that only select cells can accept the plasmid, and have a multiplecloning site (MCS) so that is can be recognized by restriction enzymes (Singh and Jain, 2013). The lac operoncreates sites that manage when the histidine-tagged protein is expressed aswell as gives a site for repressor proteins to bind to (Singh and Jain, 2013). Theribosome then begins translation which covers the histidine tagged locationfrom the start codon until the stop codon (Singh and Jain 2013).

Oncetranslated, the protein must be purified (Porath, 1992).The protein must go through resin-bound matrix that contains metal ions so thatthe histidine residues can transfer electrons to form an immobilized chelatecomplex (Porath, 1992). Finally, theprotein must be eluted (Bornhorst and Falke, 2000). There are two methods toelute the polyhistidine-tagged protein (Bornhorst and Falke, 2000). One way is to increase the pH so that the imidazolering of the histidine side will become protonated and the bonds that connectthe immobilized metal ion and the histidine side chain will break (Bornhorstand Falke, 2000). This will lead to theprotein eluting from the matrix (Bornhorst and Falke, 2000).

The second way is to use free imidazole to elute theprotein (Bornhorst and Falke, 2000).The imidazole will help the histidine bind to the metal (Bornhorstand Falke, 2000).. Histidine taggingis beneficial because it identifies protein-protein interactions, but does notaffect the function of the protein that it is involved with (Kimple et al, 2013).

His-tagged proteins are easily detected and the ionsthat the residues can bind to are stable, specifically in acidic conditions (Kimple et al, 2013). The polyhistidine tag can have a range of two to tenhistidine residues, but the most commonly seen tag consists of six residues (Kimple et al, 2013). These residues are typically found on either the Nor the C terminus (Porath 1992).

Immobilized metal-affinity chromatography (IMAC) is atechnique used to purify a protein and isolate various properties of thatprotein (Kaake et al2010). Using specific tags to trackcertain characteristics of a protein is a straightforward way to analyzecertain genes and protein-protein interactions. Histidine tagging and Green FluorescentProtein (GFP) are two distinct types of peptide sequences that act as a proteintag. The purpose of these protein tags is to enable the detection andpurification of the expressed proteins and the inherent binding in which therecould be a protein-protein interaction.