Although this simple amino acid motif is commonly found throughout nature, it does not generally result in fluorescence. It is remarkable that the principle fluorophore is derived from a triplet of adjacent amino acids: the serine, tyrosine, and glycine residues at locations 65, 66, and 67 (referred to as Ser65, Tyr66, and Gly67 see Figure 2). Properties and Modifications of Aequorea victoria Green Fluorescent ProteinĪmong the most important aspects of the green fluorescent protein to appreciate is that the entire 27 kiloDalton native peptide structure is essential to the development and maintenance of its fluorescence.
The fluorescent protein technique avoids the problem of purifying, tagging, and introducing labeled proteins into cells or the task of producing specific antibodies for surface or internal antigens. Red fluorescent proteins have been isolated from other species, including coral reef organisms, and are similarly useful. Green fluorescent protein, and its mutated allelic forms, blue, cyan, and yellow fluorescent proteins are used to construct fluorescent chimeric proteins that can be expressed in living cells, tissues, and entire organisms, after transfection with the engineered vectors. The HeLa cells were co-transfected with sub-cellular localization vectors fused to cyan ( mTurquoise) and yellow ( mVenus) fluorescent protein coding sequences (Golgi complex and the nucleus, respectively), as well as the "Fruit" protein, mCherry, targeting the mitochondrial network. A similar specimen consisting of human cervical adenocarcinoma epithelial cells ( HeLa line) is depicted in Figure 1(b). The opossum kidney cortex proximal tubule epithelial cell ( OK line) presented in Figure 1(a) was transfected with a cocktail of fluorescent protein variants fused to peptide signals that mediate transport to either the nucleus (enhanced cyan fluorescent protein ECFP), the mitochondria (DsRed fluorescent protein DsRed2FP), or the microtubule network (enhanced green fluorescent protein EGFP). Illustrated in Figure 1 are two examples of multiple fluorescent protein labeling in living cells using fusion products targeted at sub-cellular (organelle) locations. With the rapid evolution of fluorescent protein technology, the utility of this genetically encoded fluorophore for a wide spectrum of applications beyond the simple tracking of tagged biomolecules in living cells is now becoming fully appreciated. More recently, fluorescent proteins from other species have been identified and isolated, resulting in further expansion of the color palette. Since these early studies, green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors that are broadly referred to as fluorescent proteins. Over the next two decades, researchers determined that aequorin and the green fluorescent protein work together in the light organs of the jellyfish to convert calcium-induced luminescent signals into the green fluorescence characteristic of the species.Īlthough the gene for green fluorescent protein was first cloned in 1992, the significant potential as a molecular probe was not realized until several years later when fusion products were used to track gene expression in bacteria and nematodes. Due to this property, the protein was eventually christened with the unceremonious name of green fluorescent protein ( GFP). During the isolation procedure, a second protein was observed that lacked the blue-emitting bioluminescent properties of aequorin, but was able to produce green fluorescence when illuminated with ultraviolet light. Osamu Shimomura and Frank Johnson, working at the Friday Harbor Laboratories of the University of Washington in 1961, first isolated a calcium-dependent bioluminescent protein from the Aequorea victoria jellyfish, which they named aequorin.
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