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Green Fluorescent Protein - What is this mystical substance and why is it so important?

By Satya Mouktika


In today’s day and age, the world of bioluminescence is vast and broad both in terms of application and research. However, a particular fluorescent protein found in the jellyfish Aequorea victoria stands out amongst many others. This is none other than the Green Fluorescent Protein (GFP). Since its discovery in the year 1961 by Osamu Shimumora, the applications of GFP have expanded throughout the field of biological and medical engineering and research, gaining quite the importance amongst researchers in the fields. 


The jellyfish Aequorea victoria exhibiting GFP


Structure and Properties 

As of today, biologists are able to genetically modify the chromophore (the molecules of the protein that can absorb light waves) to make the GFP express a rainbow of colours, as well as produce an enhanced GFP (EGFP) which is about 35 times brighter than the original. 


Some of the genetically engineered forms of GFP.


The chromophore is the part of the protein that is sensitive to a certain wavelength of light. It absorbs light at this particular wavelength (blue light) and in turn emits light of a greater wavelength (green light). This is what gives the protein its characteristic green fluorescence. 


At the time of its discovery, GFP depended on the aequorin (another fluorescent protein found in A. victoria) to excite it upon the binding of calcium ions to it. The GFP at it’s raw state is called the ‘wild type.’ This is its natural state.


A problem posed with wild type GFP was that the optimum folding temperature of the protein (the temperature at which the linear amino acid chain can fold into a complex protein is most efficient) was in the range 9°C to 12°C which is the temperature of its usual underwater habitat. This, however, limits its applications since the fluorescence will not be visible unless the protein is folded accurately. As a solution to this problem, a less temperature sensitive variant called superfolder GFP (sfGFP) was developed. 


Applications


Theoretically, the applications of GFP in research are close to endless. In 1992, cDNA for GFP was cloned and was expressed in E.coli and C.elegans for the first time. This was a major turning point. GFP has since then revolutionised biological research by enabling real-time visualization of gene expression, protein localization, and cellular processes. It can be used in tracking gene activity, studying protein interactions, monitoring cell fate in developmental studies, and serving as a reporter gene in various biological assays. For example, GFP-tagged cancer cells help researchers study tumor growth and metastasis. 


The timeline below details the developments in GFP research and its applications.


GFP timeline



References

1) Chan, K., Hei, Y., Kwong, H., & Szeto, D. (n.d.). Green Fluorescent Protein: Its Development, Protein Engineering, and Applications in Protein Research. Journal of Young Investigators. https://doi.org/10.22186/25.3.1.1

2) Gerdes, H.-H., & Kaether, C. (1996). Green fluorescent protein: applications in cell biology. FEBS Letters, 389(1), 44–47. https://doi.org/10.1016/0014-5793(96)00586-8

3) Stauber, R. H., Horie, K., Carney, P., Hudson, E. A., Tarasova, N. I., Gaitanaris, G. A., & Pavlakis, G. N. (1998). Development and Applications of Enhanced Green Fluorescent Protein Mutants. BioTechniques, 24(3), 462–471. https://doi.org/10.2144/98243rr01

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