<P> In cell and molecular biology, the GFP gene is frequently used as a reporter of expression . It has been used in modified forms to make biosensors, and many animals have been created that express GFP, which demonstrates a proof of concept that a gene can be expressed throughout a given organism, in selected organs, or in cells of interest . GFP can be introduced into animals or other species through transgenic techniques, and maintained in their genome and that of their offspring . To date, GFP has been expressed in many species, including bacteria, yeasts, fungi, fish and mammals, including in human cells . Scientists Roger Y . Tsien, Osamu Shimomura, and Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein . </P> <P> In the 1960s and 1970s, GFP, along with the separate luminescent protein aequorin (an enzyme that catalyzes the breakdown of luciferin, releasing light), was first purified from Aequorea victoria and its properties studied by Osamu Shimomura . In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca ions, inducing a blue glow . Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green . However, its utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene . The funding for this project had run out, so Prasher sent cDNA samples to several labs . The lab of Martin Chalfie expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994 . Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later . Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish . Although this near - wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, poor photostability and poor folding at 37 ° C . </P> <P> The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in Science in 1996 . One month later, the Phillips group independently reported the wild - type GFP structure in Nature Biotechnology . These crystal structures provided vital background on chromophore formation and neighboring residue interactions . Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivatives in use today . </P> <P> Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered . The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien . This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm . This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher . A 37 ° C folding efficiency (F64L) point mutant to this scaffold, yielding enhanced GFP (EGFP), was discovered in 1995 by the laboratories of Thastrup and Falkow . EGFP allowed the practical use of GFPs in mammalian cells . EGFP has an extinction coefficient (denoted ε) of 55,000 M cm . The fluorescence quantum yield (QY) of EGFP is 0.60 . The relative brightness, expressed as ε QY, is 33,000 M cm . Superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006 . </P>

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