THURSDAY, 8 JUNE 2023GFP is one of the most famous proteins in biology. Green fluorescent protein (more commonly known simply as GFP) is, unsurprisingly, a protein that glows green. It has been, and continues to be, used throughout a wide range of different fields in cell biology to understand when specific proteins are made, where they are in cells and what they interact with. However, for a protein that is used so commonly in research, it has a rather humble origin: jellyfish. So how did GFP go from relative obscurity to leading a cell biology revolution?
THE HUNT FOR GFP | The story of GFP began in the early 1960s, when Osamu Shimomura was attempting to isolate a fluorescent protein from the jellyfish Aequorea victoria that glowed green. While this may conjure up images of bright green jellyfish floating in the sea, the jellyfish are actually transparent and do not usually fluoresce. To get them to light up, they need to be touched or shocked with electricity. Even then, it is only the rim of the jellyfish bell that glows green. Considering this, the importance that GFP would come to play hardly seems obvious from its origins.
After processing many jellyfish samples, Shimomura managed to isolate a fluorescent protein that glowed... blue. Yes, that is right, blue. The initial fluorescent protein identified from Aequorea victoria was not GFP. It turned out that this jellyfish uses two fluorescent proteins to glow green when stimulated. The first protein identified was named aequorin, and it glowed blue in the presence of calcium ions. Shimomura went on to identify the second protein which would become known as GFP. He found that the wavelengths of light needed to activate GFP overlapped those emitted by aequorin. Therefore, when the jellyfish are stimulated, calcium ions enter the cells that contain aequorin, and activate it. The energy that is then emitted from aequorin is transferred to GFP, causing the GFP to emit green light.
Isolating GFP was a major step on the path to GFP becoming a cell biology revolution. However, to really have an impact, GFP needed to be expressed in cells other than those of a jellyfish.
MAKING CELLS GLOW | Before GFP could be expressed in other organisms, its gene needed to be identified. To achieve this, Douglas Prasher and colleagues created chains of DNA nucleotides based on knowledge of GFP’s amino acid sequence. They then used these to isolate DNA encoding GFP from a library of DNA that encodes all the proteins made in Aequorea victoria.
At this time in the early 1990s, it was still unclear if specific enzymes were required for GFP to become functional. This question was solved by Martin Chalfie who expressed GFP first in the bacteria Escherichia coli, and then in the microscopic worm Caenorhabditis elegans, using DNA encoding GFP from Prasher. By showing that fluorescent GFP could be produced even in bacteria, which are so distantly related to jellyfish, Chalfie demonstrated that no specific enzymes were required for GFP to function. In fact, oxygen is the only factor required for GFP to fluoresce in cells, as demonstrated by Roger Tsien when he expressed GFP in bacteria without oxygen. Subsequently, GFP would be expressed in numerous other model organisms, including yeast, fruit flies, and various mammalian cells.
MODIFYING GFP | Even though scientists could now readily express GFP in a variety of cell types, some researchers realised that they did not need to settle for the default properties of GFP. Tsien was one of the first people to start tinkering with the structure of GFP, by changing which amino acids were present at specific positions in the protein. This was possible because of the relatively stable structure of GFP, which can be modified in certain ways without losing its function. Tsien and colleagues mutated GFP to increase its brightness and to create variants that emit different colours, including blue, cyan, and yellow. The initial work by Tsien has been expanded by other researchers, leading to a variety of GFP variants with improved properties, including the ability to fold into the fluorescent form more efficiently at 37°C, and an optimised DNA sequence encoding GFP for more efficient expression in mammalian cells.
THE GFP TOOLBOX | Developing variants of GFP allowed scientists to create a toolbox of fluorescent proteins. But what is it that scientists can do with GFP and its variants that has revolutionised cell biology? After all, scientists were using fluorescent compounds bound to antibodies before GFP was discovered, which allowed them to visualise the location of proteins in cells. The significant difference is that, unlike fluorescent compounds like fluorescein, GFP is a protein, so it can be expressed by cells. This difference is crucial to the importance of GFP.
To visualise proteins by fluorescence microscopy using fluorescent antibodies, cells need to be fixed. This is where cells are treated with a compound that crosslinks the different proteins in a cell together, effectively freezing a cell in its current state. Therefore, the cell is no longer alive, and the proteins cannot move. Using this method allows researchers to identify the locations of proteins only at specific times.
That is where GFP comes in. Scientists can now create a fusion of their protein of interest and GFP, which can be done without either protein losing its function. This means that fluorescence microscopy can monitor the location of the protein of interest within cells in real time.
Scientists have taken this one step further by using a process called fluorescence resonance energy transfer (FRET), to identify if two proteins bound to different GFP variants bind to each other. If they do, energy will be passed from one GFP variant to the other, causing the second GFP to fluoresce. (This is the same process that naturally occurs between aequorin and GFP in Aequorea victoria!)
FRET has been instrumental in studying protein-protein interactions within cells. Researchers have also used GFP in other ways to investigate these interactions, such as by developing split-GFP. This is where two proteins of interest are fused to different halves of the same GFP variant. Upon the two proteins interacting, the two GFP fragments can combine into a whole GFP and fluoresce. By using multiple types of split-GFP variants in the same cell, multiple protein-protein interactions can be visualised at the same time.
GFP has even been used to create biosensors such as calcium sensors. Tsien and colleagues fused two different GFP variants to opposite ends of a calcium-binding protein. When calcium ions enter a cell, the calcium-binding protein changes shape and causes FRET between the GFP variants, allowing the change in ion concentration to be visualised.
GFP and its variants have been used in a variety of different types of experiments in addition to those above. As such, GFP has become an essential part of the cell biologist’s toolkit.
GFP’S LEGACY | Scientists have used variants of GFP across many different fields in cell biology. The wide-reaching impact of GFP was recognised by the 2008 Nobel Prize in Chemistry, which was jointly awarded to Shimomura, Chalfie, and Tsien.
GFP has had such a wide impact and is used so commonly, that it is easy to take its existence for granted these days. However, GFP would not be what it is today if it did not have the properties that make it so special and that allowed it to be developed further. Given its unusual origin in jellyfish, it may have not even been discovered in the first place!
Looking back on its story, GFP is proof that breakthroughs across science can start from basic research in niche fields.
Andrew Smith is a 4th year PhD student studying neurodegenerative disease at Christ’s College. Artwork by Sumit Sen.