Fluorescent Dyes

Structure and function of green fluorescent protein

Green fluorescent protein (GFP) is a protein composed of about 238 amino acids, which can be excited from blue light to ultraviolet light and emit green fluorescence. While many other marine organisms have similar green fluorescent proteins, traditionally, green fluorescent protein (GFP) refers to the protein that was first isolated from jellyfish Victoria. This protein was first discovered by Osamu Shimomura et al. in 1962 in jellyfish Victoria. This luminescence process also requires the help of the luminescent protein aequorin, and this luminescent protein can interact with calcium ions.

green fluorescent protein

On October 8, 2008, Japanese scientist Osamu Shimomura, American scientists Martin Chalfie and Qian Yongjian won the Nobel Prize in Chemistry that year for their discovery and modification of green fluorescent protein.

The wild-type green fluorescent protein found in Victoria multitubular luminescent jellyfish, 395nm and 475nm are the largest and second largest excitation wavelengths, respectively, and its emission wavelength peak is at 509nm, which is in the position of green light bluish in the visible spectrum . The fluorescence quantum yield (QY) of green fluorescent protein was 0.79. The green fluorescent protein obtained from sea pansy only has a higher excitation peak at 498 nm.

In cell biology and molecular biology, the green fluorescent protein (GFP) gene is often used as a reporter gene. The green fluorescent protein gene can also be cloned into vertebrates (eg: rabbits) for expression, and used to demonstrate a hypothetical experimental method. Through genetic engineering technology, the green fluorescent protein (GFP) gene can be transferred into the genomes of different species and expressed continuously in the offspring. Now, the green fluorescent protein (GFP) gene has been introduced and expressed in many species, including bacteria, yeast and other fungi, fish (eg, zebrafish), plants, flies, and even mammalian cells such as humans.

Development History

In 1962, it was reported that scientists had extracted proteins with bioluminescent properties from the luminous hydromedusan Aequorea. In the 1970s, there were some new developments in the phenomenon of bioluminescence. Some scientists have studied the intramolecular energy transfer in the bioluminescence system of the genus Aequorea. In the early 1990s, scientists cloned the GFP cDNA and studied its expressed amino acid sequence, and found that the gfp 10 cDNA encodes a 238 amino acid peptide segment. The clone of A. victoria GFP gene was studied, and it was found that there were three restriction enzyme cleavage sites on the GFP gene. This is of great help for subsequent scientists to understand its structure.
In February 1994, M. Chalfie et al. creatively expressed GFP in Escherichia coli and Caenorhabditis elegans cells respectively, and concluded that since GFP luminescence does not require other substrates or co-factors, the expression of GFP can be used for Monitor gene expression and protein localization in vivo. In the period since then, countless researchers have devoted themselves to GFP-related research. In the past month or so of M. Chalfie’s report, Tsuji et al. fused and expressed the GFP protein in E. coli, and the excitation and emission spectra of GFP in the organism were not significantly different from those under natural conditions. Because the fluorescence intensity of GFP in living organisms is not strong enough, it is difficult to apply it to practical scientific research. In 1995, Tsien et al. improved the luminescence intensity of GFP, which greatly promoted the application of GFP in biological research. Then in August 1996, F. Yang et al. analyzed the molecular structure of GFP. The GFP protein is barrel-shaped, with 11 β-sheets forming a periphery, with an α-helix inside, and the ends of the barrel are Some irregular curls. In September of the same year, Tsien et al. analyzed the crystal structure of GFP and clarified its luminescence principle. There are also scientists who create mutants to screen for better GFPs, such as pH-sensitive GFPs, GFPs specifically for plant cell research, and so on. In addition to optimizing GFP, many scientists have developed their thinking and extended the application of GFP protein to many research fields. In 2002, David A. Zacharias and others applied GFP protein to the study of membrane proteins. In the same year, the GFP protein was even made into a Zn biodetector.


Wild-type green fluorescent protein starts out as a 238 amino acid peptide chain, about 25KDa. Then according to certain rules, 11 β-sheets form a cylindrical fence around the outer periphery; in the cylinder, the α-helix holds the chromophore almost exactly in the center. The chromophore is surrounded by the center, which can avoid dipolarized water molecules, paramagnetic oxygen molecules, or cis-trans isomerism and chromophores, resulting in fluorescence quenching.

Fluorescence is the most special feature of fluorescent proteins, and the chromophore plays a major role. The 65, 66, and 67 amino acids on the α-helix – serine, tyrosine, and glycine undergo cyclization, dehydrogenation, etc. to form a chromophore. Interestingly, the chromophore formation process is catalyzed by residues on the peripheral fence, and the substrate requires only oxygen. This suggests the potential of GFP to be widely used in different species: it can be independently expressed as a functional protein in different species without the need for additional factors. However, the exact process is still being discussed.
The conjugated π bond on the chromophore can absorb the excitation light energy, and after a short time, release the energy in the longer wavelength emission light, resulting in fluorescence.


Because fluorescent proteins can be stably inherited in future generations and can be specifically expressed according to promoters, they have gradually replaced traditional chemical dyes in quantitative or other experiments. More, fluorescent proteins have been transformed into different new tools, which not only provide new ideas for solving problems, but also may bring more valuable new problems.

Fluorescence microscope
Main article: Fluorescence microscopy
The availability of GFP and its derivatives has completely redefined fluorescence microscopy, and the way it is used in cell biology and other biological disciplines. Among them, the most exciting is for super-resolution microscopy imaging.