Green Fluorescent Protein, or GFP, is a protein that glows bright green when exposed to blue or ultraviolet light, a characteristic that has made it a widespread tool in biology and medicine. It allows researchers to observe biological systems non-invasively, illuminating structures and processes that were once invisible. This ability to image a wide range of proteins and their functions has had a substantial impact on cell biology.
The Discovery and Origin of GFP
The story of GFP begins with the jellyfish Aequorea victoria off the west coast of North America. In the early 1960s, scientist Osamu Shimomura was studying its bioluminescence to understand how the jellyfish produced light. During his research, he isolated a protein he named aequorin, which emits a blue light in the presence of calcium ions.
As a byproduct of his research, Shimomura also discovered GFP. He observed that while aequorin produced blue light, the jellyfish itself glowed green. He later demonstrated that GFP absorbs the blue light from aequorin and re-emits it as green light. This foundational work, involving an estimated 850,000 jellyfish, earned Shimomura a share of the 2008 Nobel Prize in Chemistry, alongside Martin Chalfie and Roger Y. Tsien.
Shimomura’s focus was on basic chemistry, and he did not intend to find a marker for medical research. Other scientists recognized its potential, however. Douglas Prasher first cloned and sequenced the GFP gene in 1992, paving the way for its use as a tool. Martin Chalfie later successfully expressed the gene in other organisms, demonstrating its utility as a marker.
How GFP Works as a Biological Marker
GFP’s function as a biological marker is based on its use as a “reporter gene.” Scientists use genetic engineering to fuse the gene that codes for GFP with the gene of a protein they wish to study. This combined genetic information is then introduced into a cell. When the cell produces the target protein, it also produces an attached GFP molecule, tagging the protein with a fluorescent beacon.
This process is effective because GFP is a self-contained system. The protein has 238 amino acids, and a small sequence of three of these spontaneously folds into a structure called a chromophore. This chromophore is what absorbs high-energy light and emits it at a lower-energy, green wavelength. Unlike many other bioluminescent proteins, GFP does not require additional enzymes or cofactors to become fluorescent, only oxygen.
Because the GFP tag is genetically encoded, it can be passed to subsequent generations of cells for long-term studies. The fluorescence is visualized using a fluorescence microscope, allowing researchers to see the location, movement, and concentration of their protein of interest in real-time. This has been likened to placing a tiny, glowing light bulb on a specific molecule to watch where it goes and what it does.
This technique also allows for monitoring gene expression. By placing the GFP gene under the control of a specific promoter—a region of DNA that initiates transcription—scientists can determine when and in which cells that promoter is active. If the cells light up, it indicates that the promoter is “on” and the associated gene is being expressed.
Visualizing Biological Processes with GFP
The ability to tag proteins with GFP allows researchers to move beyond static images and observe complex processes as they unfold in living systems. This has provided insights into fields ranging from neurobiology to cancer research.
In neuroscience, GFP has been used to watch the development of nerve cells in the brain. By tagging neuronal proteins, scientists can observe how neurons grow, form connections (synapses), and create intricate circuits. This visualization is important for understanding both normal brain development and the progression of neurodegenerative diseases like Alzheimer’s. Researchers can literally watch as nerve cell damage occurs over time.
Cancer research has also been advanced by GFP. Scientists label cancer cells with the protein to track their movement and spread throughout a living organism, allowing for real-time visualization of metastasis. This is the process by which cancer spreads from its primary site to other parts of the body. Observing how cancer cells invade tissues and form new tumors helps researchers understand the disease and test new anti-cancer drugs.
GFP is also used to study how cells respond to infections by tagging viral proteins to watch the life cycle of a virus. Developmental biologists use GFP to trace the fate of cells in a growing embryo, mapping how different tissues and organs form from a single fertilized egg. In these applications, GFP acts as a guiding light, making the invisible world of the cell visible.
Expanding the Genetic Color Palette
The utility of the original green fluorescent protein was expanded by the work of Roger Tsien. He recognized that to study multiple biological processes at once, scientists would need more than one color. Tsien began to modify the amino acid sequence of the original GFP, creating variants that fluoresced in colors like blue (BFP), cyan (CFP), and yellow (YFP).
His team’s efforts to create a red fluorescent protein (RFP) from jellyfish GFP were challenging, so the solution came from coral. By isolating a red fluorescent protein from a coral species, Tsien’s team completed the primary color spectrum. This palette of proteins, often named after fruits like strawberry and cherry, allows researchers to tag multiple proteins or cells with different colors.
The development of this multicolor toolkit led to techniques like “Brainbow.” In this method, researchers use genetic engineering to randomly express combinations of different fluorescent proteins in individual neurons. This results in each neuron being labeled with one of up to 100 different colors, creating a detailed map of the brain’s wiring. By tracing these distinctly colored neurons, scientists can untangle the complex circuits that underlie thought and behavior.
This ability to color-code cellular components has broad applications. For example, researchers can visualize the real-time interactions between cancer cells (labeled red) and host immune cells (labeled green). This allows for direct observation of how the immune system responds to a tumor, providing a more dynamic view of the microscopic world.