Protein As the result, a purified protein

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). 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).

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

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 is 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 result of the expression of GFP in influenza viruses, influenza vaccines were brought to clinical trials (Bestebroer et al., 2011).

In conclusion, both his-tags and GFPs can cloned into a strand of DNA. This strand of DNA (with the inserted gene) can be used to further purify a protein or

 

 

The neutral protonated or A form absorbs in the

ultraviolet (UV) at about 395 nm, whereas the anionic

or B form absorbs at about 475 nm.

The GFP chromophore can exist as anionic or neu-

tral, which explains the spectroscopic observation of

two coincident absorbance and excitation bands Fig.

3(b). The neutral protonated or A form absorbs in the

ultraviolet (UV) at about 395 nm, whereas the anionic

or B form absorbs at about 475 nm.

The GFP chromophore can exist as anionic or neu-

tral, which explains the spectroscopic observation of

two coincident absorbance and excitation bands Fig.

3(b). The neutral protonated or A form absorbs in the

ultraviolet (UV) at about 395 nm, whereas the anionic

or B form absorbs at about 475 nm.  (Giannone, 2014).