Literature Review: Green Fluorescent Protein And Histidine Tagged Proteins

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Literature Review: Green Fluorescent Protein and Histidine Tagged Proteins Histidine and green fluorescent protein (GFP) tagging are important in molecular biology because they allow for purification, tracking, and quantification of target proteins (Ferrari et al., 2004; Cho et al., 2011; Deponte, 2012). Histidine tagging provides a method for isolating and increasing the amount of target protein recovered from a biological organism or mixed sample of proteins (Masek et al., 2011; Singh and Jain, 2013). GFP tagging allows target proteins to be tracked, quantified, and observed within a living organism (Deponte, 2012). Histidine tagging works by attaching a specific number of histidines to the carboxyl or amino terminus of a target protein …show more content…
This allows the histidines to bind to the metal ion matrix filter while everything else in the mixture is eluted out (Bornhorst and Falke, 2010; Singh and Jain, 2013). To optimize IMAC, a DNA sequence coding for 6 histidines is most commonly used because it gives the highest affinity for the metal ions while not disturbing the function of the target proteins (Singh and Jain, 2013). The metal ions used in the filter are zinc, cobalt, nickel, or copper, due to histidines high binding affinity for these ions (Darain et al., 2004; Bornhorst and Falke, 2010). After the rest of the mixture has been washed out of the IMAC column and disposed of, a chemical wash buffer is used to displace the histidine tagged proteins from the matrix surface, allowing them to elute out by changing the binding affinity histidine has for the metal ions (Bornhorst and Falke, 2010). To optimize the elution of the target proteins from the matrix surface, imidazole is most commonly used because lower pH values may denature or affect the function of the proteins (Reichel et al., 2007; Bornhorst and Falke, 2013). Histidine tagging is very useful in molecular biology because it allows target proteins to be purified and specifically selected from a mixture of biological molecules and proteins (Darain et al., 2004; …show more content…
This can provide information about the way proteins move through cells, interact with other proteins, and implement their effects inside living cells (Stretton et al., 1998; Scott et al., 1998). Flow cytometry, specifically FACS when dealing with GFP tagged proteins, is a way to measure and sort proteins and molecules from a mixed biological sample based on their chemical and physical make-up (Ferrari et al., 2004). FACS is a quick and easy method to separate target proteins and molecules by passing the mixture through a stream of continuously running water that quickly splits the stream into individual droplets containing the cells (Ferrari et al., 2004). The droplets pass through a fluorescence detection system that can measure the fluorescence of a single cell, or more appropriately, the amount of proteins in a cell that are giving off fluorescence (Ferrari et al., 2004). Therefore, the proteins that have been tagged with GFP are able to be quantified and sorted, providing a purified sample of the cells containing the proteins of interest, as well as quantifying the amount of protein present in each cell (Ferrari et al., 2004). FACS, in combination with GFP tagging, can be a very useful tool for rapid identification and purification of target proteins (Scott et al., 1998; Stretton et al.,

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