Green Fluorescent Protein GFP

Green fluorescent protein (GFP) is also a photoprotein isolated and cloned from the jellyfish Aequorea victoria. Variants have also been isolated from the sea pansy Renilla reniformis. GFP, like aequorin, produces a blue fluorescent signal, but without the required addition of an exogenous substrate. 

All that is required is an ultraviolet light source to activate the fluorescent properties of the photoprotein. This ability to autofluoresce makes GFP highly desirable in biosensing assays since it can be used on-line and in real-time to monitor intact, living cells. Additionally, the ability to alter GFP to produce light emissions besides blue (i.e., cyan, red, and yellow) allows it to be used as a multianalyte detector. 

Consequently, GFP has been used extensively in bioreporter constructs within bacterial, yeast, nematode, plant, and mammalian hosts (Fig. 4). Some examples of GFP applications in mammalian cell systems, where its use has revolutionized much of what we understand about the dynamics of cytoplasmic, cytoskeletal, and organellar proteins and their intracellular interactions. Some other applications of GFP are monitoring tumor cells in gene therapy protocols, identification of HIV in infected cells and tissue, monitoring of protein-protein interactions in living cells etc. 

FIGURE 4 Assembly of a bioluminescent bioreporter integrated circuit (BBIC) sensor. 

Chemistry and Physics of Complex Materials Concepts and Applications 

Fluorescence emission maximum Fluorescence quantum yield Molar absorption coefficient (e) at peak wavelengths (as monomer) 

It is generally believed that the absorption (and fluorescence excitation) peak at about 400 nm is caused by the neutral form of the chro-mophore, 5-(p-hydroxybenzylidene)imidazolin-4-one, and the one in the 450-500 nm region by the phenol anion of the chromophore that can resonate with the quinoid form, as shown below (R1 and R2 represent peptide chains). However, the emission of light takes place always from the excited anionic form, even if the excitation is done with the neutral form chromophore. This must be due to the protein environment that facilitates the ionization of the phenol group of the chromophore. This is also consistent with the fact that the pACa values of phenols in excited state are in an acidic range, between 3 and 5 (Becker, 1969), thus favoring anionic forms at neutral pH. 

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Quantum yield of luciferin. Various values of quantum yield have been reported for coelenterazine in the luminescence reaction catalyzed by Renilla luciferase 0.055 (Matthews et al., 1977a), 0.07 (Hart, et al., 1979), and 0.10-0.11 (with a recombinant form Inouye and Shimomura, 1997). The quantum yield is significantly increased in the presence of Renilla green fluorescent protein (GFP) see below. 

Gonzalez, D. S., Sawyer, A., and Ward, W. W. (1997). Spectral perturbations of mutants of recombinant Aequorea victoria green-fluorescent protein (GFP). Photochem. Photobiol. 65 21S. 

Kojima, S., et al. (1998). Fluorescent properties of model chromophores of tyrosine-66 substitute mutants of Aequorea green fluorescent protein (GFP). Tetrahedron Lett. 39 5239-5242. 

Several studies were performed on the optimization of expression levels of ELP proteins in E. coli. In a recent example, the expression protocol was optimized for an ELP fusion with green fluorescent protein (GFP). This fusion protein was expressed and purified in a yield of 1.6 g/L of bacterial culture, which finally yielded 400 mg GFP/L bacterial culture. This extremely high yield was found after uninduced expression in nutrient-rich medium supplemented with phosphate, glycerol and certain amino acids, such as proline and alanine [234]. The influence of fusion order was also examined and it was found that positioning the ELP at the C-terminus of target protein resulted in significantly higher expression levels [35]. 

Chia Normally it is a basal crescent during mitosis, but Yuh-Nung Jan s lab has shown that in insc mutants, green fluorescent protein (GFP)—PON can form cortical crescents but these are not fixed to the basal cortex and can move. 

FIGURE 1.2 In situ localization of the OCP-green fluorescence protein (GFP) fusion protein Immunogold labeling of a thin section of OCP-GFP transformed Synechocystis PCC6803 OCP-GFP cells were labeled with a polyclonal antibody against the GFP coupled to 10 nm gold particles. Bar = 0.5 pm. 

Stoner-Ma D, Melief EH, Nappa J et al (2006) Proton relay reaction in green fluorescent protein (GFP) polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP. J Phys Chem B 110 22009-22018... 

Shimomura O (2009) Discovery of green fluorescent protein (GFP) (Nobel lecture). Angew Chem Int Ed Engl 48 5590-5602... 

Bublitz G, King BA, Boxer SG (1998) Electronic structure of the chromophore in green fluorescent protein (GFP). J Am Chem Soc 120 9370-9371... 

Seebacher C, Deeg FW, Brauchle C, Wiehler J, Steipe B (1999) Stable low-temperature photoproducts and hole burning of green fluorescent protein (GFP). J Phys Chem B 103 7728-7732... 

Wiehler J, Jung G, Seebacher C, Zumbusch A, Steipe B (2003) Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP). Chembiochem 4 1164—1171... 

The discovery of Green Fluorescent Protein (GFP) and the development of technology that allows specific proteins to be tagged with GFP has fundamentally altered the types of question that can be asked using cell biological methods. It is now possible not only to study where a protein is within a cell, but also feasible to study the precise dynamics of protein movement within living cells. We have exploited these technical developments and applied them to the study of translation initiation factors in yeast, focusing particularly on the... 

Since about the mid-1990s, the green fluorescent protein (GFP), and its spectral variants [1] have become some of the most exciting molecules in microscopy, biochemistry, and cell biology (see Chapter 5). 

RicinB fused to green fluorescent protein (GFP) Tobacco leaf and hairy root culture GFP-specific IgG present in serum IgA in semm and fecal matter. 10... 

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