▎WuXi AppTec Content Team Editor
The 2022 Nobel Prize in Physiology or Medicine will be announced soon,
“For the past decade, green fluorescent protein has served as a beacon, showing the way to countless biochemists, biologists, medical scientists and other researchers.”
——2008, the official news of the Nobel Prize
▲The 2008 Nobel Prize in Chemistry was shared by three scientists, Osamu Shimomura, Martin Chalfie and Roger Y. Tsien, who Made important contributions to the discovery and development of the green fluorescent protein GFP (Image source: The Nobel Foundation)
100,000 glowing jellyfish
In the early summer of 1961, Osamu Shimomura, 33, drove 5,000 kilometers with his newly married wife to Friday Harbor on San Juan Island, Washington. It is calm and peaceful, with beautiful scenery. The rocks on the coast are scattered with colorful sea urchins and starfish of different sizes. The grouper caught and made into sashimi are also very delicious.
However, this is not a honeymoon for newlyweds. As a postdoc at Princeton University, Shimomura is trying to solve a problem: Why does the glowing jellyfish (Aequorea victoria) glow?
▲Quiet Friday Harbor (Image source: Reference [3])
These jellyfish are abundant in Friday Harbor, and when they are stimulated, the outer edge of the jellyfish glows with a green ring. Throughout the summer, Shimomura, his wife and colleagues continued to fish for the glowing jellyfish, cut the edges of the jellyfish one by one, then grinded and filtered them, trying to extract the glowing substances.
However, the experiment did not go well at the beginning. Shimomura tried various conditions, but still could not find the expected luminescent substance.
Just as clueless, Shimomura poured the jellyfish extract into the sink and saw a bright blue flash. Due to a leak in the aquarium, some seawater flowed into the tank. Osamu Shimomura realized that something in the sea water was triggering the glowing reaction. The speculation was soon confirmed by experiments that calcium ions in seawater were necessary for light emission.
Having this key clue, over the next few days, Shimomura and the head of his lab, Frank Johnson, finally identified the glowing substance from 100,000 glowing jellyfish The active ingredient was named aequorin. When this protein encounters a very small amount of calcium ions, it emits blue light and is the first light-emitting protein ever discovered by humans.
▲The color of a glowing jellyfish (Aequorea victoria) in sunlight and when stimulated in a dark room (Image source: Reference [3])
In their paper published in 1962, they described the discovery of aequorin in detail, and mentioned, “by the way”, that at the same time they discovered and purified another much less abundant “Green protein” – later green fluorescent protein (GFP).
Compared to aequorin, the structure of GFP appears to be “simple”. It consists of 238 amino acids strung together into a long chain that bends and folds into a barrel-like structure. The three amino acids located in the “barrel” interact to form a special chromophore. The chromophore of GFP absorbs the blue light emitted by aequorin and then emits green light, which is why the luminescent jellyfish emits green light instead of blue light.
Spiritual “light” appears
GFPs have existed in glowing jellyfish for more than 160 million years, and their light will illuminate 21st-century biology. However, they were discovered in Yushimomura After that, GFP will have to wait another 30 years before it starts to shine.
In 1989, Martin Chalfie of Columbia University first heard about aequorin and green presence of fluorescent proteins. This made his eyes light up, and he immediately recorded the knowledge of fluorescent proteins in the notebook.
It is worth mentioning that Martin’s early academic career may resonate with some graduate students. He initially had no idea what he was going to study. He did research on anti-tumor drugs and tried The research on modified amino acids failed to produce decent results in the end, and they simply gave up and ran to study social sciences.
▲Martin and his first guitar, he once said that if he had no scientific achievements, he might become a guitarist (Image source: Reference [6])
After quitting the lab, he also worked as a middle school teacher, but returned to the lab after just one year of teaching and felt inappropriate. In order to successfully produce his graduation thesis, Martin changed from this laboratory to that laboratory again. With the help of his roommate, he learned to cultivate C. elegans, which also paved the way for the application of GFP.
One of the characteristics of nematodes is that they are transparent, so if the green fluorescent protein is put into the worm’s body, not only can you see where the light is shining, but it can also be used as a marking tool.
According to his vision, linking genes encoding GFP with genes encoding other proteins, or with promoters (equivalent to “gene switches”) that control other genes, would Green fluorescence can be used as an indicator to see where other proteins are made, or when a gene switch in a cell is turned on.
The idea is great, but to make it happen, the first step is to find the GFP gene in the genome of the glowing jellyfish, and then isolate it, so-called “cloning” the gene.
Martin heard that Douglas Prasher of the Woods Hole Oceanographic Institution was working on cloning the GFP gene and had a similar idea of using GFP to tag the protein. After exchanges, the two decided to cooperate.
A few years later, Douglas successfully completed the cloning of the GFP gene, and he graciously sent the plasmid containing the gene to Martin and many interested scientists.
▲Escherichia coli expressing GFP (Image source: Reference [4])
As Martin had been expecting, when he and his colleagues managed to put a fragment of the gene encoding GFP into E. coli cells, they saw that under UV light, E. coli emits exciting green fluorescence! This pivotal experiment demonstrated that the chromophore of the GFP protein can be formed spontaneously in E. coli cells without any other cofactors.
Next, Martin tried a new experiment on the worms he was familiar with, using GFP as a marker of gene expression. Using DNA technology, he placed the gene encoding GFP behind a promoter that is active only in the six neurons responsible for touch.
▲Nematodes expressing GFP (Image source: Reference [4])
The experiment was successful again! A transparent nematode with several nerve cells glowing green appeared on the cover of the February 1994 issue of Science.
▲The February 1994 issue of “Science” reported the results of Martin and his collaborators’ GFP experiments in the form of a cover article (Image source: Reference [4], Science Screenshot of the official website)
Martin’s experimental results quickly aroused the interest of many people, and a technological innovation with GFP as the core appeared.
From fluorescent green to kaleidoscope
Like Martin, UCSD moneyDr. Roger Tsien also received a plasmid with the GFP gene from Douglas.
Dr. Yongjian Qian specializes in applying chemistry to solve biological problems. But this became a problem in job applications, because the biology department thought he was a chemist, and the chemistry department refused him to study biological problems.
Fortunately, this interdisciplinary interest eventually gave the GFP revolution a giant leap forward.
After in-depth study of the chemical structure of the GFP chromophore, Dr. Qian Yongjian modified the amino acid composition of GFP with the help of DNA technology. Through random mutation, he and his colleagues developed several new versions of green fluorescent protein, including enhanced GFP, which emits more intense light, and blue, cyan and yellow fluorescent proteins.
▲Escherichia coli expressing four GFP mutants and emitting different colors (Image source: Reference [2])
Since then, a laboratory in Russia, looking for GFP-like proteins in coral polyps, found a red-emitting protein called DsRED. Once again, Dr. Qian Yongjian led the research team to modify DsRED to make this protein more stable and more suitable for connecting with other proteins, which can enter cells like GFP as a fluorescent marker for biological processes.
Now there are more and more colors on the palette. On the basis of the modified version of DsRED, Qian Yongjian’s research group developed a series of colored fluorescent proteins, which were named plum (mPlum), cherry (mCherry) according to the color of their light. , strawberry (mStrawberry), orange (mOrange), etc.
▲Colorful fluorescent protein (Image source: Reference [5])
These colorful colors make biological processes that are otherwise invisible to the naked eye suddenly clear and vivid. The fluorescent proteins illuminated growing tumors, exposed the whereabouts of disease-causing bacteria, and revealed nerve cells that were being injured by Alzheimer’s disease.
Using these fluorescent proteins, scientists have for the first time seen how virus particles assemble in living cells, how nerve cells in the brain form dense neural networks through nerve fibers, and The movement of individual proteins within cells, as well as their interactions with other proteins, can be tracked.
In addition to this, GFP has a role in broader biotechnological applications. For example, genetically engineered bacteria that can glow green in the presence of toxic metals can be used to test water quality.
▲Using three fluorescent proteins (yellow, cyan and red), scientists turned the mouse brain into a kaleidoscope (Image source: Reference [1])
In August 2016, Dr. Qian Yongjian, the creator of green fluorescent protein, passed away suddenly. In October 2018, Dr. Osamu Shimomura, who discovered green fluorescent protein, also passed away. The light they left behind will never be forgotten.
References:
[1] The Nobel Prize in Chemistry 2008. Retrieved Sep 23th, 2022 from https:https://www.nobelprize.org/prizes/chemistry/2008/press-release/
[2] The green fluorescent protein: discovery, expression and development. Retrieved Sep 23th, 2022 from https:https://www.nobelprize.org/uploads/2018/06/advanced- chemistryprize2008-1.pdf
[3] Osamu Shimomura: DISCOVERY OF GREEN FLUORESCENT PROTEIN, GFP https:https://www.nobelprize.org/uploads/2018/06/shimomura_lecture.pdf
[4] Martin et al., (1994) Green fluorescent protein as a marker for gene expression. Science. DOI: 10.1126/science.8303295
[5] Nathan C Shaner et al., (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein Nature Biotech. DOI: 10.1038/nbt1037
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[6]Martin Chalfie Biographical. RetrievedSep 23th, 2022 fromhttps:https://www.nobelprize.org/prizes/chemistry/2008/chalfie/biographical/
