GFP is got from Aequorea Victoria a jelly fish which has a photoprotein Aequoin which absorbs light and transfers to an the intact fluorescent protein, GFP where the efficiency of the light is increased and greater wavelength light is emitted. GFP has 238 amino acid and has a 11-Î² stranded barrel like a cylinder and the Î± chain runs around it and forms a lid on top called "Î² can ". Chromophore lies in center inside the Î² can. Changes in the covalent structure and the stereochemistry of the flurophore changes the obsorption and emission spectral of the wtGFP making it Novel for various studies. (Prasher, Eckenrode et al. 1992).
Fluorescence is due to the crystallisation of the tripeptides ser65, Tyr66, Gly67 and 1,2-dehydrogenation that leads to the formation of p-parahydroxybenzylideneimidazolelinone it is a aerobic reaction. The folding and the oxidation is a long process and it is not of biochemical interest. The protein is resistant to extreme conditions of pH upto 11 after which it loses its absorbance. It fluorescence upto 65C, resistance to protease enzyme and also chemical detergents like 1%SDS(Cubitt, Woollenweber et al. 1998). GFPs are created which are not acid sensitive. This can be made advantages because if the light is quenched in a particular organal we can know the pH of the organel by knowing the sensitivity of the mutant.(Tsien 1998).
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WtGFP has a maximum absorption at 397nm and a minimum at 475nm due to the neutral phenols and the anionic phenolates and the emission is at 507. The extinction co-efficient of GFP was found to be 21-30mM-1 cm-1 and the quantum yield 0.72-0.85. when their product was calculated which gives the brightness it was found that the brightness is low compared to the free fluorescein. There are other problems of photoisomerization and photobleaching.
Photoisomerization leads to decrease in the absorption of 395nm and increase of absorption in the 495nm this will lead to decrease in intensity of fluorescence at 395nm illumination. Photobleaching leads to decrease in absorption of both the wavelength(Cubitt, Woollenweber et al. 1998). After the cDNA of GFP was found mutants have been created to minimise these problems. Mutagenesis was done to the tripeptide and other aminoacid sequence to solve problems of aggregation, maturation problems at alleviated temperature, increase in intensity and energy transfer, extinction co-efficient, spectral variations and also photobleaching. one of such mutant is Photoactivation GFP (paGFP). Photoactivation is created where there is intense illumination and the fluorophore undergoes photoconservation and increases the minor peak absorption. This leads to three fold increase in the fluorescence. paGFP has increased optical enhancement under aerobic condition s making it suitable to mark protein and cell population(Patterson and Lippincott-Schwartz 2002). Enhanced GFP is created by shifting the absorption equilibrium toward the anionic side and it lead to an increase the photostability, brightness, single wavelength emission and also the photostability. Mutation in Ser65T led to a good absorption of 470-490nm and emission of 510nm and there was a red-shift in the absorbance 489 and the maturation was faster, the extinction-coefficient six times higher and it is more resistant to photobleaching. Emission wavelength greater than red was not got due to the intrinc primary structure of GFP or the fluorescence property of GFP.(Remington 2000). Mutation in T66H and T145F also gives a variant fluorescent protein called Blue fluorescent protein( BFP). We can study the localization and expression of more than one proteins by more spectral variant in the cell and then analysing them invivo with FRET for any protein- protein interaction. Another interesting type is the Reversible switchable fluorescent protein (RSFP) here, due to irradiation of a specific light wavelength there may be fluorescence or no-fluorescence. This is because of the change in the cis-trans conformation of the chromophore in its protonation state (Stiel, Andresen et al. 2010) (Cubitt, Woollenweber et al. 1998; Patterson and Lippincott-Schwartz 2002; Zimmer 2009).
GFP due to its sensitivity, non-destructive monitoring of the gene and heterologous expression without the requirement of any exogenous substrate or cofactors can be used as a reporter gene. It can be used as a reporter gene in various forms. By fussion tag where a chimeric protein is formed (GFP + endogenous protein) and then the fluorescent is studied
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(Tsien 1998). Dicistronic expression can also be done where the GFP and the gene of interest is under the control of the same promoter. This reduces the screening process as the cell that expresses the GFP also expresses the protein of interest.(McLachlin, Cornetta et al. 1990).
GFP is autocatalytic and can express continually in host cell. It does not create toxicity or enter into the mammalian cell. It also does not involve in the cellular activity of the pathogen and so GFP can be introduced into the pathogen and its effect can be studied by analysing in the epifluorescence microscope. Pathogens can also be quantitated with flow cytometry either alone or in association with macrophages(Valdivia, Hromockyj et al. 1996; Valdivia and Ramakrishnan 2000). It can be used in biosensors for detecting ions (Tsien 1998).