Green+Fluorescent+Protein

Daniel Labuz

**Origins of Green Fluorescent**
Green fluorescent protein (GFP) was discovered in a jellyfish, //Aequorea victoria//. The GFP was isolated and extracted from the jellyfish in 1962 by a number of researchers (Shimomura et al.1962). It wasn’t until 1994 that there was a breakthrough in GFP research that led to the current uses of today (Chalfie et al. 1994). Chalfie et al., (1994) established a method to monitor gene expression in //E. coli// and //Caenorhabditis elegans// (Figure 1). This was a seminal study in GFP usage and provides many useful techniques for this protein. ========

**Green Fluorescent Protein Technique and Purpose**
The mechanism of how GFP works is illustrated in Figure 2. There are many proton transfers when GFP is exposed to ultraviolet light that produces a green color that is visible by the naked eye. The purpose of GFP in nature is still debated but it’s possible that these fluorescence proteins protect organisms from UV radiation (Salih et al., 2000).

The two main approaches for GFP as a technique are biosensors and gene expression (Palmer et al., 2011). GFP in gene expression is easily done by chemical means, and many undergraduate students will perform this experiment in genetics or biochemistry laboratory (Figure 3).



GFP as a biosensor is relatively new technique within the last decade. A simple example of this is tagging a target enzyme with GFP and monitor it’s action. This can be referred to as FRET or fluorescence resonance energy transfer (Niino et al. 2009). Also, GFP can be used as a micro pH reader. Since the proton transfers happen at specific pH monitoring the color of the GFP protein can give clues about the pH of a specific compartment (Palmer et al., 2011 & Remington, 2011). Another popular biosensor is the use of GFP as a micro redox center (Remington, 2011, Cannon & Remington, 2008). This technique is advantageous because one is able to monitor a specific pathway without any intrusions or harmful modifications.

**Research Using Green Fluorescent Protein**
As said before GFP is used in many fashions in research as biosensors and for gene expression. Niino et al. (2009) used different colored GFP to label cardiac myocytes and visualize them in real time. Using a GFP label in this case was essential because these cells are extremely motile and tracing them would be difficult without fluorescence. Additionally, this GFP label was able to monitor calcium and cAMP levels by the intensity of fluorescence.

In a study by Vermeer et al. (2009), the authors used a modified lipid binding GFP to monitor the activity of Phosphatidylinositol 4-phosphate. Polyphosphoinositides are important in eukaryotic cells for signaling and trafficking events in the cell, even though they are not very abundant. GFP was used as a harmless tracking device in this study, making the protein very useful in many applications. The study found that this lipid is important for the growth of new root tips on plants. Without the help of GFP the authors would not be able to use a living host to visualize the plant growing, thus they would be unable to make this conclusion.

Lastly, for a good amount of time researchers have been trying to come up with a GFP that consist of red shades. Shcherbo et al. (2009) have recently made a pH stable, non-toxic, and photostable GFP protein that reaches into the red color spectrum. This opens up new doors for those that want to label proteins and cells in living hosts. As mentioned before, being able to label within a living host is essential to tracing a pathway or a protein in it’s natural state and having multiple GFP colors will let you accomplish what Niino et al. (2009) did by using four different colors.

**References**
Bent DV, Hayon E (1975) Excited state chemistry of aromatic amino acids and related peptides. I. Tyrosine. Journal of American Chemical Society. 97:2599–2606.

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802-805.

Labuz DR, Osorio D, Alsanius B, Windstam S (2012) Mechanically damaged baby greens and the effect on Escherichia coli O157-H7 growth. 2nd Annual Summer Scholars Poster Conference. Oswego, NY. Sept. 2012.

Niino Y, Hotta K, Ola K (2009) Simultaneous live cell imaging using dual FRET sensors with a single excitation light. PLoS ONE 4. Palmer AE, Qin Y, Park JG, McCombs JE (2011) Design and application of genetically encoded biosensors. Trends in Biotechnology. 29:144–152.

Remington JS (2011) Green fluorescent protein: A perspective. Protein Science. 20:1509-1519.

Salih A, Larkum A, Cox G, Kuehl M, Hoegh-Guldberg O (2000) Fluorescent pigments in corals are photoprotective. Nature 408:850–853.

Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM (2009) Far-red fluorescent tags for protein imaging in living tissues. The Biochemical Journal. 418: 567–574.

Shimomura O, Johnson FH, Saiga Y (1962).Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. // Journal of Cellular // and Comparative // Physiology //. 59:223–239.

Tolbert LM, Solntsev KM (2002) Excited-state proton transfer: from constrained systems to ‘‘super’’ photoacids to superfast proton transfer. Accounts of Chemical Research 35:19–27.

Vermeer JEM, Thole JM, Goedhart J, Nielsen E, Munnik T, Gadella TWJ (2009) Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells. The Plant Journal. 57: 356–372