Immobilized Green Fluorescent Protein (GFP) are two distinct

Immobilized metal-affinity chromatography (IMAC) is a
technique used to purify a protein and isolate various properties of that
protein. Using specific tags to track certain characteristics of a protein is a
straightforward way to analyze certain genes and protein-protein interactions. Histidine
tagging and Green Fluorescent Protein (GFP) are two distinct types of peptide
sequences that act as a protein tag. The purpose of these protein tags is to enable
the detection and purification of the expressed proteins and the latent binding
in which there could be a protein-protein interaction.

Histidine tagging is a method of IMAC that
is very simple to use and can achieve a significant increase of purification in
a single step. The polyhistidine residue must first be fused to the gene of
interest within a plasmid. The plasmid must be capable of replication, contain
a gene available for the allowance that only select cells can accept the
plasmid, and have a multiple cloning site (MCS) so that is can be recognized by
restriction enzymes (  ). The lac operon
creates sites that manage when the histidine-tagged protein is expressed as
well as gives a site for repressor proteins to bind to. The ribosome then
begins translation which covers the histidine tagged location from the start
codon until the stop codon. Once translated, the protein must be purified. The
protein must go through resin-bound matrix that contains metal ions so that the
histidine residues can transfer electrons to form a immobilized chelate complex
(  ). Finally the protein must be eluted.
There are two methods to elute the polyhistidine-tagged protein. One way is to increase
the pH so that the imidazole ring of the histidine side will become protonated
and the bonds that connect the immobilized metal ion and the histidine side chain
will break. This will lead to the protein eluting from the matrix. The second way
is to use free imidazole to elute the protein. The imidazole will help the histidine
bind to the metal. Histidine tagging is beneficial because it identifies protein-protein
interactions, but does not affect the function of the protein that it is
involved with. His-tagged proteins are easily detected and the ions that the
residues can bind to are stable, specifically in acidic conditions. The polyhistidine
tag can have a range of two to ten histidine residues, but the most commonly
seen tag consists of six residues. These residues are typically found on either
the N or the C terminus.

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The green fluorescent protein (GFP) is a bioluminescent
protein that was first discovered within Aequorea
victoria, which is a species of jellyfish. The green fluorescent protein
works be producing a fluorescent glow in vivo allowing scientists to study the
dynamics of the protein under a microscopy without altering or killing the cell
( ). Within a plasmid, GFP can be placed subsequent to the promoter region and beside
the gene of interest. The restriction enzymes can cut the section of interest
out and then add the enzyme ligase and then the plasmid can be added to a
protein ( ).  Within a protein, GFP can
be fused to either the N or C terminus or between two proteins to study the
protein-protein interactions but the insertion must be done strategically so
there is a lack of disruption to the natural reading frame ( ). Due to its fluorescence,
scientists can isolate where the gene is expressed and when GFP is triggered by
blue ultraviolet light and produces a green fluorescent light. When a light
with a wavelength of 400nm strikes the chromophore, the protein goes into an
excited state. When the chromophore enters ground state, the light emitted will
be at the wavelength 505nm. This light appears on the visible light spectrum as
a green colour.  Unfortunately, there
have been instances in which there are cellular side effects in regards to the
GFP expression such as apoptosis within the cell lines.

In research done by _____, human breast cancer cells
were analyzed using green fluorescent proteins to track proteome changes. Breast
cancer cells were cultured and treated with antibiotics under restricted
conditions and were then tagged with GFP. The proteins were extracted after
grown to a set size and analyzed using two-dimensional electrophoresis and isobaric
tags for relative and absolute quantitation (iTRAQ).  Comparisons were made on cell behaviour and
protein-protein interactions between the cells that were treated with
antibiotics and tagged with GFP and the breast cancer cells that went untreated
( ). It was found that the antibiotic improved the outcome of the cells, but
many results came up inconclusive. There was proof of analytical variability and
evidence of molecular changes caused by the GFP expression within the cell ( ).

Ultimately,
both green florescent proteins tagging and polyhistidine tagging are both
useful to monitor and track specific gene activity. Both have their pros and
cons as methods of immobilized metal-affinity chromatography, but with further
research there can be improvements in both techniques to improve the tracking
of genes and protein-protein interactions over time.