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Protein tagging is of great
importance in molecular biology research and experiments (Chase and Kuo,
2010). In research, two
protein tags, specifically Histidine (His-tag) and Green Fluorescent Protein
(GFP), are used to study the function, cellular pathways, and interactions
between proteins (Murayama
and Kobayashi 2014). As protein tagging is employed
more frequently, the knowledge on molecular and cellular biology concepts
continue to expand.

His-tags or
polyhistidine tags, are amino acid chains comprising of approximately six
histidine residues incorporated into the N or C terminus of a target protein (Chase
and Kuo, 2010). The His-tag is expressed in a vector, and fused in frame of the
protein of interest to facilitate protein purification (Chase and Kuo, 2010).

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Protein purification allows for the study of the structure and function of a
protein (Ghahremanzadeh
et al., 2017). Understanding the structure
and function aids to determine diagnostic and therapeutic applications for a
target protein (Ghahremanzadeh
et al., 2017). Purifying a protein can
be done via immobilized metal affinity chromatography (IMAC) (Chase and Kuo, 2010).

IMAC is a technique that exploits histidine residues by separating them from His-tagged
proteins (Ghahremanzadeh
et al., 2017). Histidine residues have
a high affinity for metal ions such as Cu2+, Co2+, Zn2+,
and Ni2+ (Barbosa
et al., 2015). (say why) On the charged resin
of the IMAC column, immobilized Nickle ions (Ni2+) form strong interactions
with the histidine residues of the his-tag (Ghahremanzadeh et
al., 2017). Ni(II)-nitrilotriacetic acid (Ni-NTA) is a metal-chelating
agent that aids to chelate the histidine residues to nickel ions of the IMAC
resin (Loughran and Walls, 2014). High concentrations of imidazole are added to
the column to prevent non-specific protein binding to the IMAC resin (Chase and
Kuo, 2010). This allows the six histidine residues to separate from the recombinant
protein and remain bound to the Ni-NTA chelate group (Loughran and Walls,
2014). As the result, a purified protein can be eluted from the column (Loughran
and Walls, 2014).

The primary
structure of GFP is a long polypeptide chain of 238 amino acids (Remington,
2011). When arranged into the tertiary structure, GFP forms an eleven stranded -barrel
with an -helix
inside (Remington, 2011). Three amino acids: tyrosine 66, serine 65, and
glycine 67 are centered within the -helix
(Remington, 2011). In the presence of oxygen, the amino acids get oxidized and
forms a mature GFP chromophore (Remington, 2011). For the fluorescence of the
matured GFP chromophore, it must exist in one of two forms: the neutral
protonated/A form or the anionic/B form (Remington, 2011). The A form absorbs
ultraviolet light (UV) at approximately 395 nm whereas the B form absorbs light
in the blue range (approximately 475 nm) (Remington, 2011). As a fluorescent
tag, GFP has advanced studies in protein localization, gene expression, and
protein-protein interactions (Murayama
and Kobayashi 2014).

A protein of interest can be fused with a 714 base pair gfp at its N- or C- termini to visualize the target proteins
interactions in real time (Murayama
and Kobayashi
2014). Talk about other colors.

 

 

 

 

 

 

 

In 2009, a
pandemic outbreak known as swine flu (H1N1 and H3N2 influenza virus) infected a
large proportion of the human population (Bestebroer et al.,
2011). Therefore, the need for an influenza vaccine was at an all-time high to cease
the ever-growing human cases (Bestebroer
et al., 2011). Recombinant
influenza viruses were constructed to carry the swine subtype, H1N1 (Bestebroer et al., 2011). Upon infection, reverse
genetic plasmids were used to clone the GFP gene in the neuraminidase (NA) gene
at the 3′-5′ termini (Bestebroer
et al., 2011). Various animal
species were experimentally infected with the GFP-expressing viruses and a
fluorescence imager was used to track the localization within the specimen (Bestebroer et al., 2011). Titers were
obtained from the specimen infected with the GFP-expressed virus strains (Bestebroer et al., 2011). It was discovered
that antibodies directed to the H1N1 and H3N2 influenza viruses (Bestebroer et al., 2011). Thus, as a results
of the expression of GFP in influenza viruses, influenza vaccines were brought
to clinical trials (Bestebroer
et al., 2011).

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