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Green fluorescent protein (GFP) is a fluorescent molecule that is found in the jellyfish Aequorea victoria. Isolated in 1961 and cloned in 1992, GFP has become a popular reporter molecule for gene expression in both mammalian and nonmammalian systems. Some modifications of GFP have resulted in the creation of an important class of reporter molecules which provides superior monitoring capabilities to those of other reporters such as luciferase or alkaline phosphatase. GFP can also be used as a nontoxic reporter in whole organisms.
Properties of the GFP
The GFP, first so named by Morin and Hastings, contains 238 amino acids. Residues 65-67 (Ser-Tyr-Gly) in the GFP sequence spontaneously form the fluorescent chromophore p-hydroxybenzylidene-imidazolinone (Figure 1).
Figure 1. The chromophore is a p-hydroxybenzylidene-imidazolidone (green background). The cyclized backbone of the residues forms the imidazolidone ring. The peptide backbone trace is shown in red.
The excitation spectrum of GFP fluorescence has a dominant maximum at around 400 nm and a significantly smaller maximum at around 470 nm, while the emission spectrum has a sharp maximum at about 505 nm and a shoulder around 540 nm (Figure 2).
Figure 2. Fluorescence excitation (full-line curve) and emission (dashed curve) spectra of native GFP from Aequorea Victoria.
The crystal structure of GFP is an eleven-stranded β-barrel, threaded by an α-helix, running up along the axis of the cylinder. The chromophore is in the α-helix, close to the center of the can-like cylinder (Figure 3). A large part of the primary structure of the protein is used to construct the threading α-helix and the β-barrel. The N-terminal residue and the C-terminal residues 230-238, approximately corresponding to the maximal numbers of residues which can be removed from the N-(2 residues) and C-terminal (6 residues) respectively of GFP at retained fluorescence, are disordered and therefore unresolved in this structural image.
Figure 3. The tertiary structure of GFP.
Modifications of the GFP
To improve detection in mammalian systems, researchers have modified the sequence of GFP. One common variant, enhanced green fluorescent protein (EGFP), contains two mutations in the chromophore region-Phe64 to Leu and Ser65 to Thr- that red-shift the spectral excitation peak to 489 nm. Red-shifted GFPs, such as EGFP, can be excited by the 488 nm argon-ion laser most commonly found in flow cytometry equipment and can be detected by standard fluorescein (FITC; fluorescein isothiocyanate) filter sets used in both fluorescence microscopy and flow cytometry. Longer-wavelength excitation also minimizes photoconversion of the chromophore.
The generation of red-shifted GFP variants only hinted at the apparent tolerance of the chromophore to mutagenesis. Because of the creation of red-shifted GFPs, further amino acid manipulations within the chromophore region have successfully produced color shifts into the yellow and blue regions of the visible spectrum (Figure 4). Yellow fluorescent protein (YFP), originally published as the 10C mutant, emits a yellow-green fluorescence that is distinguishable from GFP in flow cytometry and confocal microscopy, and is detectable by standard FITC filter sets. Human codon optimization of YFP, in conjunction with additional chromophore-associated mutations, has resulted in the creation of YFP variants that often surpass the fluorescence intensities of GFP in mammalian cells. Other amino acid changes have resulted in the creation of cyan fluorescent proteins (CFPs) and blue fluorescent proteins (BFPs).
Figure 4. (a) Excitation and (b) emission spectra of EGFP and its color variants.
However, the color palette obtained by mutating GFP is still insufficient, and researchers go back to nature to find more fluorescent proteins. Until now, over 150 distinct fluorescent or colored GFP-like proteins have been reported. GFP-like fluorescent proteins (FPs) have been found in marine organisms ranging from chordates (e.g., amphioxus) to cnidarians (e.g., sea pansies and corals). No FPs have been found in terrestrial organisms. The majority of GFP containing organisms are non-bioluminescent. These fluorescent proteins can be divided into seven groups on the basis of their color and chromophore structure (Figure 5).
Figure 5. Fluorescent proteins. E stands for enhanced versions of GFP, m is monomeric proteins, and tdTomato is a head-to-tail dimer.
Applications of the GFP
Green fluorescent protein (GFP) has changed from a nearly unknown protein to a commonly used molecular imaging tool in biology, genetics, chemistry and medicine. GFPs and GFP-like proteins (i.e., chromoproteins and fluorescent proteins) are particularly useful due to their stability, and the fact that the chromophore is formed in an autocatalytic cyclization of the 65SYG67 sequence, which does not require a cofactor. This means that unlike most other bioluminescent reporters, GFP fluoresces in the absence of any other proteins, cofactors, or substrates. Therefore, GFP can be used as a genetic tracer molecule. Furthermore, it appears that fusion of GFP to a protein does not change the location or function of the protein.
GFP can be introduced into mammalian cells or whole organisms either by traditional plasmid transfection or by the viral infection. Retroviral, adenoviral and other viral infection systems employing GFP are all being developed for facilitating gene therapy studies. Because the GFP gene is relatively small, it can be efficiently integrated into expression vectors without substantially increasing the vector’s size. This feature allows vectors to be constructed that contain both a gene of interest, for example, a therapeutic gene, and the GFP gene for use as a marker, without losing infection or transfection efficiencies. In many cases, use of GFPs as a marker for efficient integration provides an improvement over more traditional antibiotic selection. Successfully transfected cells can be quickly sorted for GFP fluorescence by flow cytometry for immediate therapeutic use.
GFP can also be used to mark successful transgenics, particularly because it is not toxic to the transgenic animal and can be monitored quickly and noninvasively by illumination by near-UV or blue light. GFP can be fused to a gene or tissue-specific promoter. And in transgenic animals, expression can be monitored when a given tissue is subjected to certain promoter-responsive stimuli. Because GFPs can be viewed noninvasively, they may prove valuable tools for detecting tumors or diseased tissues in whole animals, or in human patients. GFPs are ideal for use in areas of research ranging from drug discovery to agricultural engineering. Potential uses for GFPs increase, and we expect to see more novel applications for GFPs in the future.