It is difficult to create a road without a road: the legendary discovery journey of green fluorescent protein

Green fluorescent protein can form chromophores by self-catalysis and emit green fluorescence under the stimulation of blue light or ultraviolet light. Through genetic engineering and fusion with other proteins, it can make invisible proteins visible. Therefore, in the past two decades, it has become a guide for biologists and medical scientists to study various biochemical processes in cells. It can be said to be an important tool for biological research. The original discovery and subsequent important development of green fluorescent protein won the 2008 Nobel Prize in Chemistry, and the exploration journey of the scientists involved can be called a legendary story in the history of science.

Written by | Xu Yixun

The development of the history of life sciences is from the macroscopic level that is easy to observe (such as species classification and gross anatomy) to the microscopic level that requires instruments to observe (such as tissues and cells studied in microscopic anatomy). In the 17th century, Dutch scientist Antonie van Leeuwenhoek first observed and described single-celled organisms with his improved optical microscope, which was a watershed in the history of biology. With the help of microscopes, biologists gradually observed microscopic research objects such as bacteria, cells, and organelles that were previously unknown. Once such reductionist research reaches the molecular level, even electron microscopes make it difficult for us to directly observe the expression and localization of biological macromolecules such as proteins in living cells. The green fluorescent protein (GFP) isolated from the Victoria multi-tube luminous jellyfish (Aequorea victoria, hereinafter referred to as luminous jellyfish or jellyfish) makes the once invisible protein visible. In the past two decades, it has become a guiding star for biologists and medical scientists to study various biochemical processes in cells. This article tells the stories of several scientists who have made important contributions to the biological revolution triggered by GFP.

Early studies of bioluminescence

The discovery of GFP is closely related to the phenomenon of bioluminescence, so we must first introduce the different types of low-temperature luminescence.

Figure 1: Various types of low-temperature luminescence phenomena and their excitation modes

Fire is the most important invention in human history. Incandescence, which is associated with it, is usually defined as visible light emitted when an object is heated to a high temperature. Low-temperature luminescence is a type of visible light spontaneously emitted by excited chemical components that are not in thermal equilibrium with the environment. More than 2,500 years ago, the ancient Greek scientist Aristotle wrote in his book On Colors: "Some objects that are not fire and have nothing to do with the production of fire seem to emit light naturally." This means that humans have long been aware of the important difference between incandescence and low-temperature luminescence: incandescent bulbs are not efficient in lighting, and can only convert a small part of electrical energy into light energy, while the rest of the energy is dissipated in the form of heat; while bioluminescence is an efficient chemical reaction that produces almost no heat in the process of converting chemical energy into light energy, so it is also called "cold light."

According to different excitation modes, we can classify low-temperature luminescence into many types, such as photoluminescence, electroluminescence, chemiluminescence (bioluminescence is a special type of chemiluminescence), etc. (Figure 1). The most common photoluminescence is fluorescence and phosphorescence. Readers are advised to pay special attention to the difference between fluorescence and bioluminescence.

The most common bioluminescent phenomenon in nature is fireflies. Every summer night, fireflies dance in the grass, creating a magical scene. Li Bai, a great poet in the Tang Dynasty, once wrote a poem called "Ode to Fireflies": "Rainstorms are hard to extinguish, and the wind makes them brighter. If they fly up to the sky, they will become stars beside the moon."

Figure 2: The study of bioluminescence began with scientists’ fascination with the phenomenon of fireflies.

In addition to fireflies, there are many species in nature that have the ability to emit light at low temperatures, including bacteria, protozoa, fungi, jellyfish, squid, etc. Scientists have long been curious about the phenomenon of bioluminescence, but have always lacked effective scientific research methods. It was not until 1667 that British chemist Robert Boyle used an air pump to remove the air from the bell jar and found that the fungi inside no longer glowed. When he introduced air again, the fungus's bioluminescence ability was restored. In the 17th century, people in the chemical world knew nothing about the composition of air. It was not until the 1770s that Swedish chemist Carl Wilhelm Scheele and British chemist Joseph Priestley independently discovered oxygen, which was eventually explained by French chemist Antoine Lavoisier, that the dependence of bioluminescence on oxygen finally surfaced.

After more than a century of stagnation, the exploration of the chemical mechanism of bioluminescence was brought to a new turning point by French physiology professor Raphael Dubois. In an experiment in 1885, Dubois first homogenized the luminous tissue of the click beetle (Pyrophorus) in a test tube with cold water and found that the extract dimmed after a short period of luminescence. The tissue extract he obtained with boiling water did not glow at all. To his surprise, when the cooled hot water extract was added to the cold water extract that had stopped luminescence, the mixture actually glowed again (Figure 3). If Dubois wanted the cold water extract to continue to glow, he needed to continuously add cooled hot water extract.

Figure 3: Dubois’ famous experiment in 1885 in which he first discovered the principle of bioluminescence of “luciferin-luciferase” [Pieribone, V. & Gruber, DF (2005) Aglow in the Dark: The Revolutionary Science of Biofluorescence, Belknap Harvard.]

Dubois subsequently obtained similar experimental results in other bioluminescent organisms, including fireflies, and he came to two important conclusions: (1) In addition to oxygen, the bioluminescent reaction requires at least two chemical components; (2) The "fuel" component in the luminescent reaction can withstand the high temperature of boiling water, while the "igniter" or catalyst is not heat-resistant. Dubois decided to borrow the Latin word Lucifer (literally "messenger of light") from Roman mythology to name the two components: the heat-resistant catalyst was named luciferase, and the heat-resistant small molecule was named luciferin (French: luciferine, English: luciferin).

Subsequent studies by many biologists have shown that: for many luminescent species, luciferases have different protein sequences, and luciferins also present diverse organic small molecule structures, but the "luciferin-luciferase" bioluminescence principle is valid. The goal of bioluminescence researchers can also be specific, and they can select a luminescent species of interest and use biochemical methods to separate and purify different luciferins and luciferases. Through in-depth research on the firefly luminescence system, scientists soon discovered that in addition to oxygen, luciferin, and luciferase, ATP and Mg2+ ions are also necessary conditions (Figure 3).

Bioluminescence is not common for terrestrial species, but more than 90% of marine organisms in the deep sea can emit bioluminescence. The intensity of sunlight decreases 10 times for every 75 meters below sea level. Below the depth where sunlight cannot reach, bioluminescent animals have obvious advantages in finding food, escaping enemies and attracting mates. After roughly explaining the bioluminescence mechanism of fireflies, many scientists turned their attention to marine bioluminescent organisms, the most famous of which is Professor E. Newton Harvey, who founded the school of bioluminescence at Princeton University in the United States.

In 1916, Harvey, then 28 years old, went on a honeymoon trip to Japan with his wife. The sea near the Misaki Rinkai Experiment Station was suitable for swimming at night. While swimming, Harvey became fascinated by a luminous marine organism called Vargula hilgendorfii (formerly known as Cypridina). After being collected and drained, the sea firefly can be preserved for a long time, and can glow again after being wetted with water. Therefore, it was regarded by Harvey as the best experimental material for studying bioluminescence using biochemical methods. Harvey's laboratory found that the luminescence system of the sea firefly is simpler than that of the firefly. It only needs luciferin, luciferase, and oxygen, but does not require ATP and Mg2+ ions (Figure 4). However, after partially purifying the luciferin of the sea firefly, the Harvey team worked hard for more than 20 years and could not obtain its crystals. Without high-purity luciferin, they could not deeply study the chemical mechanism of sea firefly luminescence by determining its molecular structure.

Figure 4: Luciferin-luciferase bioluminescence system of the sea firefly

Osamu Shimomura crystallizes and purifies luciferin from firefly

The difficulty in completely purifying the luciferin of sea fireflies provided an opportunity for Osamu Shimomura, the first protagonist of the GFP story, to step onto the historical stage. Compared with James Watson, who was also born in 1928, Shimomura's "starting line of life" was simply the "negative control" of the latter, full of ups and downs. Because his father was a soldier, Shimomura was mainly raised by his grandmother who lived in Isahaya City, Nagasaki Prefecture. In April 1941, Shimomura, who had just entered the first grade of Isahaya Junior High School, and his classmates had to participate in military training in accordance with the "National General Mobilization Law" revised by the Japanese government in March of that year. After entering the third grade in the fall of 1944, the school began to cancel classes frequently and required students to go to a military aircraft repair factory in Omura City for voluntary labor. The US military soon set its sights on this military factory and dispatched more than 20 B-29 bombers to completely destroy it. Several of Shimomura's classmates who did not run fast enough were unfortunately killed.

As the saying goes, "Good fortune never comes alone, and misfortune never comes singly." At 10:57 a.m. on August 9, 1945, Nagasaki was hit by the second atomic bomb of the U.S. military. At that time, Osamu Shimomura and several classmates were working in another military factory 15 kilometers away from the center of Nagasaki. When the familiar air raid alarm sounded, they walked out of the factory and climbed a nearby hill to watch. Osamu Shimomura saw a B-29 bomber flying south to the city center and dropping three cargo parachutes. Later, they learned that these were three radio high-altitude sounders dropped by the "Great Artist" aircraft during the interval between the atomic bomb drop and the final explosion that they did not see. At this time, everyone mistakenly thought that the bombing might not be a big threat, so they decided to return to the factory and try to continue working. As soon as they sat down, the strong flash outside the window temporarily blinded the students for half a minute, followed by a loud bang and a sudden change in air pressure... The distance between the military factory and Nagasaki was obviously the key to Osamu Shimomura and his friends' survival.

Although World War II ended with Japan's surrender, Shimomura Osamu, who was only 17 years old, still could not see any hope for the future. Many teachers and students of Isahaya Junior High School died in the atomic bomb explosion, and all student files were destroyed, so the junior high school students in recent years could not graduate normally. Shimomura Osamu applied for high school (Japanese: Gaode Gakkou) or technical college (Japanese: Gaode Gongye Gakkou) for two consecutive years, but failed because he could not provide junior high school grades. It was not until April 1948 that the teachers and students of Nagasaki Medical College suffered heavy casualties in the atomic bomb explosion. The urgent need for reconstruction led Shimomura Osamu to be admitted to the School of Pharmacy, which he was not interested in (Figure 5). This was also his only opportunity to receive higher education at that time.

Figure 5: Temporary campus of Nagasaki Pharmaceutical College in 1948

The teaching resources of the Nagasaki Medical College, which had just been rebuilt on the ruins, were extremely scarce. Twelve of the original 20 professors died in the atomic bomb explosion, and four were seriously injured. Most of the teaching tasks of the School of Pharmacy courses could only be undertaken by inexperienced lecturers. Due to the limitation of teaching funds, Shimomura Osamu mainly trained in analytical chemistry and physical chemistry during his three years of undergraduate studies, and had only a few opportunities to learn organic chemistry knowledge or conduct organic synthesis experiments. Shimomura Osamu's analytical chemistry teacher Shungo Yasunaga soon discovered the student's outstanding hands-on ability and specially allowed him to take some reagents home to study separation and purification using capillary chromatography. This research eventually led to Shimomura Osamu publishing the first paper of his academic career in Japanese in 1953, jointly with Professor Yasunaga.

In March 1951, Shimomura Osamu graduated from Nagasaki Pharmaceutical College with the highest total score in the class and applied for employment at Takeda Pharmaceutical Company, but an interviewer frankly pointed out that his personality was not suitable for development in a corporate environment. Professor Yasunaga helped Shimomura Osamu in time and invited him to stay on campus as an assistant teacher of analytical chemistry. Shimomura Osamu did not deliberately plan his future life. As long as he had a job, he would concentrate on doing it. He never thought about applying for graduate school to obtain a higher degree. After Shimomura Osamu worked for four years, Professor Yasunaga secured an opportunity for him to visit other institutions for one year with pay.

As the first benefactor in Shimomura's career, Professor Yasunaga also took the initiative to help him find a suitable visiting laboratory. Yasunaga's connections in the Japanese chemical industry are mainly at Nagoya University. He believes that Professor Fujio Egami, who specializes in biochemistry, is the best candidate to broaden Shimomura's scientific research horizons. Japan's telephone communication system has not been fully restored for many years after the war. Professor Yasunaga can only personally take Shimomura Osamu to Nagoya by train for more than ten hours from Nagasaki. Unexpectedly, Professor Egami was away for academic conferences during those days and he did not meet him. There are many examples of accidental events changing the trajectory of historical development. If Shimomura Osamu had met Professor Egami and successfully entered his laboratory that day, readers would probably not be reading this interesting story now. The two turned to visit organic chemist Professor Yoshimasa Hirata. After a brief conversation of a few minutes, Professor Hirata welcomed Shimomura Osamu to his laboratory as a visiting student at any time.

In April 1955, Professor Hirata pointed to a vacuum dryer and said to Shimomura Osamu who had just come to the laboratory: "There are a lot of dried sea fireflies in here. This marine animal emits light through the interaction of luciferin and luciferase. The luciferin of sea fireflies is very unstable and will degrade when it encounters oxygen. Are you willing to try to purify and crystallize this luciferin?" Shimomura Osamu knew that this difficult topic was not suitable for Professor Hirata's graduate students. As a visiting student, he had no burden of studying for a degree and was determined to try it boldly with a relaxed attitude of a "newborn calf". As early as 1935, Anderson (Rubert Anderson) of Harvey Laboratory invented a two-step extraction method that could purify this very unstable luciferin part by about 2,000 times, and inferred the amino acid components in its molecular structure through absorption spectrum. Based on this, Shimomura Osamu calculated that in order to obtain luciferin of crystalline purity, at least 500 grams of dried sea fireflies would be needed as the starting material, which was 10 times the amount used in Harvey's laboratory, so he needed to build a huge Soxhlet extractor (Figure 6, left).

Figure 6: After ten months of hard work, Shimomura Osamu completed the purification and crystallization of luciferin from the sea firefly in 1956 (the black and white photo cannot show the actual deep red color of the crystals)

During his arduous scientific research, Shimomura Osamu found that nitrogen or inert gas was not enough to eliminate the consumption of fluorescein by trace oxygen in the extraction system. He had to introduce hydrogen into the system so that trace oxygen would be converted into liquid water and absorbed by sulfuric acid. Every chemist knows that improper handling of hydrogen can cause explosions, so other members of Hirata's laboratory kept a considerable distance from Shimomura Osamu during his intense experiments. Although the use of hydrogen brought Shimomura Osamu a breakthrough, various attempts to crystallize always failed. Each attempt to prepare the extract before crystallization required him to work continuously for a week with very little sleep, and the failed extract could only be analyzed for some simple components before being scrapped. Shimomura Osamu, who never gave up, worked hard like this for an average of one week every month, until one night in February 1956, when it seemed that he would face another failure. Before going home, he decided to add an equal volume of concentrated hydrochloric acid to the extract that was about to be scrapped, and after the yellow solution turned dark red, he put it on the laboratory bench overnight, preparing to try to detect which amino acids were in it the next day.

When Shimomura Osamu returned to the laboratory in the morning, he found that the solution had changed from dark red to colorless. He first thought that it was the result of hydrochloric acid causing the hydrolysis of luciferin. He then found a small amount of black precipitate at the bottom of the test tube. Looking closely under a microscope, he found that it was actually red needle-shaped crystals (Figure 6 right)! These crystals can emit light after being mixed with the luciferase extract of fireflies, officially announcing the successful crystallization of luciferin. Looking back, concentrated hydrochloric acid can only be an accidental discovery when its structure is unknown. In addition, the gas furnace in Hirata's laboratory was turned off that night, and the solution was left overnight as the room temperature continued to drop, which also helped the crystallization process.

"Heaven will not let down those who work hard." Shimomura Osamu's ten-month scientific research efforts made an unexpected breakthrough. His academic visit to Nagoya University was also extended by Professor Hirata for one year to ensure that his first English academic paper was successfully published in 1957. Frank Johnson, the successor of Professor Harvey, was already a full professor at Princeton University in the United States. After reading this paper, he could not help but marvel that a problem that had troubled the Harvey School for more than 20 years was solved by a young Japanese scholar with only a bachelor's degree! This rare success brought Shimomura Osamu an important opportunity in his career: in the spring of 1959, he received an invitation letter from Professor Johnson shortly after returning to Nagasaki Pharmaceutical College, and was scheduled to go to Princeton University as a visiting scholar for three years in the fall of the following year.

Osamu Shimomura's Inseparable Bond with Bioluminescent Jellyfish and Friday Harbor

Shimomura arrived at Princeton University in September 1960. Professor Johnson told him that the laboratory was most interested in studying bioluminescent jellyfish and hoped that he could make a breakthrough in the study of the mechanism of jellyfish bioluminescence by taking advantage of the success of studying the bioluminescence of sea fireflies (Figure 7). In order to obtain sufficient experimental materials, there was only the Friday Harbor area in the San Juan Islands of Washington State in the United States at that time, where a large number of jellyfish could be caught every summer.

Figure 7 Adapted from: Chalfie, M. (2008) Nobel Lecture.

Since 1961, Johnson and other key members of the research team have brought their own equipment and driven for seven days from Princeton to Friday Harbor to collect jellyfish almost every summer. In order to study the luminescence of jellyfish using biochemical methods, they first had to manually cut a large number of salvaged jellyfish umbrella membranes (Figure 8), and use the laboratory conditions of the local Washington University branch to freeze and preserve the squeezed liquid (squeezates) of the light-emitting organs at the edge of the umbrella membrane.

Figure 8: Aequorea Victoria distributed on the west coast of North America and the bioluminescent organs at the edge of its umbrella. Source: https://www.nobelprize.org/uploads/2018/06/popular-chemistryprize2008.pdf

The first week of research for Osamu Shimomura and Johnson on the island was not smooth. They followed Dubois's "luciferin-luciferase" idea and could not separate the two heat-resistant and heat-intolerant components. At this time, the young Osamu Shimomura felt that he did not need to stick to Dubois's theory and could separate the luminous substances of jellyfish without any assumptions. However, Johnson was unwilling to give up Dubois's theory and change his thinking because it had been repeatedly verified in many luminous species over the years. The teacher and student insisted on their own opinions and could only work at the two ends of an experimental table, and the atmosphere was quite tense and awkward.

Whenever Shimomura encountered obstacles in his research, he liked to pause his experiments and find a quiet place to meditate on new ideas. In Friday Harbor, you can go to those secluded waters with no one by boat. Shimomura rowed out to sea for several days in a row, and then lay alone in the boat with his eyes closed to think. He fell asleep several times when the boat drifted with the wind and waves. One afternoon, when Shimomura woke up from a nap in the boat, he suddenly had an inspiration: even if the bioluminescence of jellyfish has nothing to do with luciferin and luciferase, it is likely that protein is still needed. The activity of protein is sensitive to pH. Can the luminescence of jellyfish be reversibly inhibited by adjusting the pH value of the solution? Shimomura was very excited at this time. He rowed hard back to the laboratory and prepared several buffer solutions with different pH values. When the pH values ​​were 7, 6, and 5, the jellyfish extract could still emit weak light; when the pH value was adjusted to 4, the weak light of the solution disappeared, indicating that acidity can inhibit luminescent substances! When he adjusted the pH value back to neutral with sodium bicarbonate, the weak light reappeared, indicating that the inhibitory effect of acidity is indeed reversible (Figure 9).

Figure 9 Source: Shimomura, O. (2008) Nobel Lecture

This progress made Shimomura Osamu very excited, but he was still confused about why the extract only emitted weak light. At this time, the elusive opportunity visited Shimomura Osamu's "prepared mind" for the second time in his career. On a midsummer night in 1961, Shimomura Osamu worked alone until late and was exhausted. He felt that the jellyfish extract that neutralized the acid was no longer of much use, so he poured it into the sink and called it a day. Before turning off the lights and leaving, he subconsciously looked back and was surprised to see a shining blue light coming from the sink where the extract had just been poured! Shimomura Osamu, who was good at thinking, began to analyze the reasons behind this phenomenon. On the second day, he noticed that the seawater from a nearby fish tank also flowed into the same sink, so he assumed that some substance in the seawater stimulated the weak light of the jellyfish extract into strong light. Following this idea, Shimomura Osamu used "addition and subtraction" to check the ion components with higher concentrations in the seawater one by one, and soon found that calcium ions could instantly stimulate the luminescent protein in the jellyfish extract. After witnessing Shimomura Osamu's breakthrough in discovering the role of calcium ions, Professor Johnson began to have complete confidence in his scientific research capabilities.

After understanding the role of calcium ions as a luminescent initiator, Shimomura Osamu no longer needed to adjust the pH. Instead, he only needed to add the famous calcium ion chelator EDTA to the extract to more effectively and reversibly inhibit the luminescent protein, ensuring that the target protein would not be lost due to luminescence during the further separation and purification process. By the end of August 1961, Johnson's team had collected more than 10,000 jellyfish, made a crude extract containing EDTA, and then froze it with dry ice. They brought all of them back to Princeton before starting systematic protein purification (Figure 9). A few months later, they purified two proteins: the protein with a higher concentration had a yield of about 5 mg and was named Aequorin, which is a luminescent protein that can be activated by calcium ions; another "by-product" that was eluted from the liquid chromatography column before Aequorin showed a dark green color in the sun and was named Green Protein (GP; later renamed GFP). Unexpectedly, this inconspicuous "by-product" at the time eventually became a heavyweight in the history of biological science.

With the publication of several papers on the purification of aequorin, Shimomura's three-year visit to Johnson's laboratory was fruitful. In 1963, Shimomura returned to Japan and was hired as an assistant professor of water science at Nagoya University, but two years later he realized that he would rather return to Johnson's laboratory to further study the luminescence mechanism of jellyfish. After several years of unremitting efforts, Shimomura thoroughly elucidated the luminescence mechanism of aequorin under the regulation of calcium ions (Figure 10). Apoaequorin needs to covalently bind to the small molecule cofactor coelenterazine under aerobic conditions to form a stable intermediate of aequorin with bioluminescence ability. And this covalent bond is actually a peroxide bridge, a kind of "intrinsic oxygen"! This peroxide bond can be quickly broken under the excitation of calcium ions, emitting a bright blue light while forming carbon dioxide (Figure 10).

Figure 10: Biochemical mechanism of aequorin luminescence

What is even more fascinating is that coelenterazine has obvious similarities in chemical structure with the luciferin of fireflies (the work that made Osamu Shimomura famous). It is actually an "intrinsic luciferin" (Figure 11). At this time, Osamu Shimomura suddenly realized why he had hit a wall in the summer of 1961 when he followed the traditional Dubois theory to study jellyfish. This is really "the end point is back to the beginning, and I just realized it now." When we study this interesting scientific history, we can't help but think of the famous geneticist Theodosius Dobzhansky's famous saying: "Without the light of evolution, everything in biology will be incomprehensible" (Nothing in Biology Makes Sense Except in the Light of Evolution). From fireflies that require five components to glow (luciferin, luciferase, oxygen, ATP and Mg2+ ions), to sea fireflies that only require three components (luciferin, luciferase, oxygen), and then to the luminescent protein aequorin that hides the inherent luciferin and oxygen in its molecules like a rechargeable battery, biological evolution under natural selection is truly like "eight immortals crossing the sea, each showing their magical powers"!

Figure 11: Coelenterazine, the cofactor of aequorin, is an “intrinsic luciferin”

After finding out the biochemical mechanism of aequorin luminescence, Shimomura Osamu did not forget the green protein byproduct GFP. However, the content of GFP in jellyfish is relatively low. According to his preliminary estimate, it would take hundreds of thousands of jellyfish to obtain enough raw materials to purify and crystallize GFP. Shimomura Osamu's dedication to scientific research gave him the spirit of Yugong moving mountains. In order to further study GFP, he traveled a long distance to Friday Harbor every summer, year after year, until he collected enough raw materials.

From 1962 to 1974, twelve years passed in the blink of an eye. Shimomura Osamu finally purified enough and successfully obtained green crystals of GFP in Johnson's laboratory (Figure 12). In order to further study the luminescence mechanism of GFP, Shimomura Osamu estimated that he would need to consume 100 mg of pure GFP protein, but only 20 mg of GFP could be obtained by catching more than 40,000 jellyfish every summer. So he accumulated for another five years and preliminarily identified the fluorescent chromophore of GFP in 1979 (Figures 12 and 13). In 1977, Professor Johnson, who was nearly 70 years old, decided to retire, and Princeton University had no intention of retaining Shimomura Osamu, who had limited ability to obtain scientific research funds independently. Johnson could only persuade the leaders of the Department of Biology to give Shimomura Osamu enough time to find a job and provide a temporary laboratory a few miles away from the main campus. The research work on the GFP chromophore was completed by him alone with an uncertain career future.

Figure 12: Osamu Shimomura completed the purification, crystallization, and preliminary identification of the chromophore of jellyfish GFP protein in the Johnson laboratory over a period of more than ten years.

Johnson's laboratory had not mastered protein fragment sequencing technology, and they did not actively seek collaborators in this area. Without knowing the protein sequence, Shimomura Osamu's inference of the GFP chromophore was rather rough (Figure 13), and he could not give a definitive conclusion on whether the chromophore came only from the amino acid side chains of GFP (without the need for cofactors). The breakthrough understanding that GFP contained an intrinsic fluorescent chromophore in Figure 13, which changed history, had to wait until more than a decade later in the mid-1990s.

Figure 13: The jellyfish GFP protein molecule contains an intrinsic fluorescent chromophore

In 1981, with the help of many friends in the academic community, Shimomura was finally hired as a senior researcher at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, four years after Professor Johnson retired. Since then, his research has turned to other luminous organisms and no longer involves jellyfish GFP. Here, we borrow the knowledge system many years later to summarize several important findings of Shimomura's research on jellyfish luminescence: Under the stimulation of calcium ions, aequorin in the luminous organ at the edge of the jellyfish umbrella membrane achieves bioluminescence through its luciferase activity, using the coelenterazine and peroxide bond, which is the "intrinsic oxygen", to which it is covalently bound. The light energy generated by aequorin is immediately transferred to the adjacent GFP through bioluminescence resonance energy transfer (BRET), and finally emits GFP green fluorescence visible to the naked eye (Figure 14).

Figure 14: Jellyfish use the BRET mechanism to transfer the blue bioluminescence energy of aequorin to the nearby GFP and induce green fluorescence. Source: http://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html

Molecular cloning of aequorin by Prisher

In the 1960s, when Osamu Shimomura and Johnson discovered aequorin and GFP, molecular biology was still in its infancy. If biologists wanted to study the function of a protein, they could only take the traditional "one way to Huashan": prepare a large number of samples of target species extracts, and then use biochemical methods to purify the protein. For those organisms or cell lines that can be artificially cultured in large quantities, the raw materials required for protein purification are inexhaustible. However, marine organisms such as jellyfish cannot be artificially cultivated to date, and pure proteins used in experiments require labor-intensive continuous salvage and preparation to ensure supply. Once the target species no longer appears in fixed waters due to changes in the ecological environment, the study of protein function will also come to a standstill. Fortunately, with the deciphering of the genetic code and the establishment of the central dogma of molecular biology, recombinant DNA technology came into being in the late 1970s, and the reverse transcriptase from viruses gave birth to the powerful cDNA molecular cloning technology. Once biologists are able to clone the cDNA encoding the target protein into the plasmid of Escherichia coli, they can easily obtain large amounts of pure protein by culturing the bacteria. This will not only free them from worries about basic functional research, but will also make development and application more efficient.

Professor Milton Cormier of the University of Georgia in the United States has been studying bioluminescence since the 1950s, mainly studying Renilla, sea pansy in his early years. After the publication of the groundbreaking work of Osamu Shimomura and Johnson, the Cormier laboratory also began to turn part of its research focus to jellyfish. The second protagonist of the GFP story, Douglas Prasher, came to the Cormier laboratory in 1983 to start his second round of postdoctoral training. In his previous postdoctoral laboratory, Prasher focused on bacterial genetics and successfully mastered the emerging molecular cloning technology, which was not easy at the time. In 1982, the publication of the famous experimental manual "Molecular Cloning: A Laboratory Manual" (Figure 15) strongly promoted the popularization of gene cloning technology, but many technical means, including polymerase chain reaction (PCR), had not yet been invented.

Figure 15: Cover of the first edition of the Molecular Cloning laboratory manual in 1982

Cormier hoped that the newcomer, Presher, would accept the challenge of cloning the aequorin gene. Once successful, the amount of aequorin protein produced by the laboratory through E. coli in one night would exceed the total amount they could purify by catching jellyfish in Friday Harbor throughout the summer. The total amount of mRNA that can be prepared from a single jellyfish is not high, and Presher also needs to go to Friday Harbor to collect a large number of jellyfish samples like Shimomura Osamu. In 1985, after two consecutive summers of accumulation, Presher extracted enough mRNA to construct a cDNA library. Subsequently, molecular probes designed based on known protein sequences were used to screen the cDNA library and successfully isolate 6 cDNA clones encoding aequorin, corresponding to 5 protein isoforms. After Presher expressed these gene clones in E. coli, he was unable to detect the bands corresponding to aequorin on protein gel electrophoresis for several weeks. Cormier's scientific intuition told him that the sensitivity of electrophoresis detection was not enough, so he immediately asked technician Richard McCann to help Presher design a bioluminescence detection of aequorin, and finally confirmed the success of gene cloning! Through overexpression in Escherichia coli, the price of aequorin, a calcium ion dye, dropped significantly and quickly became a commonly used experimental reagent.

With the momentum of successfully cloning the aequorin gene, Prasher finally completed two long rounds of postdoctoral training and was hired as an independent assistant researcher by the Woods Hole Oceanographic Institution (WHOI) in Massachusetts, USA in October 1987. Before officially leaving the Cormier laboratory, Prasher naturally set his next gene cloning goal: GFP. Considering that the abundance of GFP protein and mRNA is much lower than that of aequorin, Prasher needs to go to Friday Harbor every year to collect nearly 70,000 jellyfish to accumulate enough total mRNA for future GFP cloning.

Although Prasher was independent in WHOI, he was unable to recruit graduate students, postdoctoral fellows, or technicians due to limited start-up funds, so he could only fight alone to clone the jellyfish GFP gene. At the same time, he was skeptical of Shimomura Osamu's hypothesis that the GFP protein needed a cofactor to fluoresce. He imagined that once the GFP gene was obtained and expressed in E. coli, if green fluorescence could be directly observed, then the GFP gene could be fused with a gene of any species through recombinant DNA technology, and fluorescence could be used to locate the expression of protein products in cells. Based on this exciting idea, Prasher submitted several applications for scientific research funds, but most of them were rejected by the review committee. Only the American Cancer Society agreed to provide $200,000 in funding.

In early 1989, after nearly two years of hard work, Prasher screened a cDNA clone named pGFP1 in the jellyfish gene library. The plasmid contained a sequence encoding 168 amino acids. It is known that the full length of GFP protein is 238 amino acids. Prasher noticed that the 5' and 3' ends of this cDNA were incomplete. This 168 amino acid protein sequence was of great help to the Ward Laboratory (William W. Ward) with whom he collaborated. They greatly improved Shimomura Osamu's 1979 GFP chromophore work and determined that the three connected amino acid side chains (Ser65-Tyr66-Gly67) inside GFP were the molecular basis for producing green fluorescence (Figure 16). However, if Prasher wanted to apply the fluorescence of GFP as a tool for molecular positioning, he had to clone the full-length cDNA of GFP, which meant that he had to go back and build a new jellyfish cDNA library.

Figure 16 Source: Cody, CW, Prasher, DC, et al (1993) Biochemistry 32: 1212 - 1218

Chalfie and Roger Tsien created GFP labeling technology

Just as Prasher began to collect jellyfish again to build a new cDNA library, the third protagonist of the GFP story, Martin Chalfie, appeared in an unexpected way. Chalfie's laboratory at Columbia University in the United States is dedicated to studying the tactile neurobiology of Caenorhabditis elegans. On April 25, 1989, he attended the department's lecture every Tuesday at noon as usual. Paul Brehm from Tufts University introduced the luminescent proteins of various organisms. Chalfie had heard of aequorin used as a calcium dye, but it was the first time he heard of the green-light-emitting jellyfish GFP. Monomeric GFP proteins excited by ultraviolet or blue light are likely to emit fluorescence without cofactors. This feature made Chalfie, who was interested, very excited.

Although C. elegans has the natural advantage of being transparent, several commonly used gene and protein expression localization techniques at the time required lengthy sample preparation steps, and because the staining reagents needed to penetrate into the nematode body, they could not be used to directly observe living animals (Figure 17, left). If GFP, which has only 238 amino acids, could really glow, researchers could use molecular biological methods to fuse it with the nematode gene of interest, and through the GFP fluorescent label on the fusion protein, they could directly observe under a microscope which cells the gene was expressed in. The next day, Chalfie made a series of phone calls to inquire whether any scientists had successfully cloned the jellyfish GFP gene, and finally found that only Prasher of WHOI could give him the answer he wanted (Figure 17, right).

Figure 17: The transparent Caenorhabditis elegans is suitable for studying cell differentiation and function during animal development. Several gene expression localization methods before GFP technology required sample preparation and could not be used to directly observe living nematodes. After Chalfie learned about the luminescent properties of GFP from Bram's lecture, he contacted Prasher through a whole day of telephone consultation. Source: Chalfie, M. (2008) Nobel Lecture.

Chalfie and Prasher had a very good conversation on the phone. They had similar ideas about the application prospects of GFP, but their cooperation could not begin until Prasher obtained the complete cDNA clone of GFP. Considering that the average fragment of the jellyfish gene library constructed with bacterial plasmids was not large enough, Prasher decided to use λ phage to construct a new cDNA library. Two years later, he screened and obtained the λGFP10 clone containing the complete sequence encoding 238 amino acids (Figure 18). Unfortunately, Prasher was no longer in the mood to celebrate this stage of success: (1) The research funding provided by the American Cancer Society had run out, and his latest funding applications were repeatedly rejected; (2) He transferred λGFP10 to Escherichia coli for expression, but the GFP protein he obtained did not fluoresce under a microscope, which shook his previous idea that GFP could glow without cofactors or invertases; (3) WHOI colleagues had no interest in his gene cloning work, and he could not get new funding and saw no hope of passing the tenure review. Prasher decided to publish the cDNA sequence of GFP first, but the paper did not go smoothly from the beginning of submission, and it took nearly a year before it was officially published in February 1992.

Figure 18 Source: Prasher, DC, et al (1992) Gene 111: 229 - 233

Before and after the publication of the paper, Prasher tried to contact Chalfie by phone. Unfortunately, Chalfie was on academic leave at the University of Utah laboratory where his wife worked because he was newly married. Just when Prasher could not start the cooperation plan because he could not contact Chalfie, Professor Roger Tsien, the last protagonist of the GFP story, read Prasher's new paper in May 1992. Professor Tsien had hoped to study the interaction between proteins through Fluorescence Resonance Energy Transfer (FRET) since his graduate school days. For several years, he had been trying to obtain the gene encoding fluorescent protein. It is much easier to introduce the marker gene into the cells to be studied than to label the protein. Professor Tsien, who is also a caring person, could see the value of the cDNA clone in Prasher's hand at a glance. Prasher told Professor Tsien on the phone that due to the difficulty in applying for funds, he would soon leave WHOI and go to the US Department of Agriculture to work, and bid farewell to the study of GFP. Prasher was willing to share the cloning of the GFP gene immediately. Unfortunately, although there were many chemists in Professor Qian's laboratory, no one had mastered the techniques of molecular biology. He had to wait until the newly recruited postdoctoral fellow Roger Heim reported in October 1992 before he could receive and process the samples sent by Prasher. This five-month delay reversed the "plot" that almost caused Chalfie to miss GFP.

Figure 19 Source: Chalfie, M. (2008) Nobel Lecture

Chalfie returned to Columbia University before the start of the fall semester in 1992. In early September, first-year doctoral student Ghia Euskirchen wanted to do her first rotation in Chalfie's lab. Chalfie heard that Ghia had just completed her master's thesis in the School of Engineering at the university, which was related to fluorescence. He lamented that there had been no news from Prasher for three years, and he could only search for GFP-related research ideas with her through computer literature searches. Chalfie was overjoyed when he saw the complete GFP gene sequence published by Prasher at the beginning of the year, and immediately contacted Prasher by phone to restart the planned cooperation. After getting Presher's GFP clone, Chalfie noticed that because only restriction endonucleases were used in the molecular cloning process instead of PCR technology (around 1992, many American research institutes, including WHOI, did not have PCR instruments. Even in Ivy League schools such as Harvard, multiple laboratories on the same floor needed to share one), λGFP10 had extra non-coding DNA sequences from the jellyfish genome at both ends of the GFP coding sequence, including 25 extra base pairs upstream of the 5' start codon (Figure 19, upper right, marked in red). Chalfie's molecular biology intuition told him that the extra sequences at both ends might interfere with the expression of GFP in E. coli, so he instructed Jia, who was doing molecular cloning experiments for the first time, to use PCR to transfer only the GFP coding sequence to the expression plasmid with the help of senior doctoral student Xue Ding.

A few weeks later, Gia obtained many colonies containing GFP expression plasmids. Since Chalfie believed that the protein product of GFP might directly emit fluorescence, she might as well take the culture dish back to the familiar engineering college and try her luck directly with the fluorescence microscope there. On October 13, 1992, Gia's laboratory notebook (Figure 19 left) fully recorded this unexpected "Eureka moment": multiple E. coli colonies emitted beautiful green fluorescence under the microscope! Chalfie was naturally excited after seeing it. He showed off the microscope photos taken by Gia for several consecutive days (Figure 19 lower right). The experimental results clearly proved that the GFP protein can spontaneously emit green fluorescence in the cells of another species without any cofactors or invertases from jellyfish.

Figure 20: The Chalfie lab used recombinant DNA technology to link the jellyfish GFP gene to the transcription promoter of the touch-sensitive neurons of the nematode Caenorhabditis elegans, successfully demonstrating that the green fluorescence of GFP can be used to specifically mark individual cells.

Source: https://www.nobelprize.org/uploads/2018/06/popular-chemistryprize2008.pdf

After completing this experiment, Jia quickly went to another laboratory for rotation. Chalfie asked the technician to try a new experiment: first connect the GFP gene to the specific promoter of the touch sensory neurons of C. elegans, and then use microinjection to transfer the newly constructed plasmid into the gonads of mature nematodes. As long as the expression of GFP is successful, the touch sensory neurons in the next generation of larvae produced by hermaphroditic nematodes will be illuminated by green fluorescence under the microscope (Figure 20). This successful breakthrough experiment was eventually recorded in the history of science in the form of a cover article in Science magazine (Figure 21).

Figure 21 Source: Chalfie, M. (2008) Nobel Lecture

Looking back many years later, Gia's "Eureka moment" could have belonged to Prasher, and the "culprit" that made him miss it was probably the extra 25 base pairs at the 5' end of λGFP10! As a single-cell prokaryote, Escherichia coli has relatively simple gene transcription regulation, and only needs a promoter plus a regulatory sequence to achieve the effect of switching. However, the transcriptional regulation mechanism of jellyfish as a multicellular eukaryote is much more complicated, requiring the promoter to interact with multiple enhancers and regulatory sequences in the short and long range (Figure 22). When the 5' end regulatory sequences from jellyfish are carried into the plasmid of Escherichia coli, they may disrupt the promoter "context" of the bacteria, thereby interfering with the normal expression of the target gene. If Prasher could use PCR technology by finding a collaborator in 1991, the history of GFP research would be rewritten. Mr. Ye Shengtao's famous short story "Three or Five More Bushels" was included in the middle school Chinese textbook, and we can summarize the tragic story of Prasher missing out on the Nobel Prize by imitating the title here: 25 more base pairs were cloned.

Figure 22: Potential molecular biological mechanism of the 25 base pairs of Prisor polyclonal interfering with GFP expression in E. coli.

After arriving at Geim, Qian Yongjian immediately called Presher. Presher sent the GFP gene clone as promised and informed him that the Chalfie laboratory had received the clone a month ago. Qian Yongjian decided to start a healthy competition with Chalfie when he was already behind at the start. The two sides exchanged information and took the initiative to avoid each other's research direction. Although Qian Yongjian was full of hope for its application prospects after learning that Chalfie had proved that GFP could emit light independently in other organisms, with his profound organic chemistry knowledge, he still could not understand how the three amino acid side chains Ser65-Tyr66-Gly67 spontaneously cyclized to form the chromophore explained by Ward and Presher, especially the step of dehydrogenation of the carbon-carbon single bond to a double bond, which is difficult to happen without enzyme catalysis (Figure 23). Qian Yongjian could only imagine two chemical pathways: (1) two hydrogen atoms combine to form hydrogen gas and are released, which is extremely unlikely to happen in a biochemical environment; (2) an oxidant is needed to take away the two hydrogen atoms, and the only oxidant that the experimenter can directly control is oxygen in the air. Professor Qian suggested that Geim cultivate E. coli containing GFP expression plasmid in a strictly oxygen-free constant temperature shaker. They were pleasantly surprised to find that although GFP protein with normal molecular weight could be seen on the electrophoresis gel, these bacteria could not emit fluorescence. When the bacterial culture was returned to an aerobic environment for two hours, green fluorescence could be seen again. Based on this, Professor Qian gave a detailed chemical mechanism for the spontaneous formation of chromophores by GFP. The theoretical inference that hydrogen peroxide would be generated was not confirmed by other laboratories until 2006 (Figure 23, upper right).

Figure 23 Source: Tsien, RY (2008) Nobel Lecture

Professor Qian was also puzzled by the high and low double peaks in the excitation spectrum of wild-type GFP. Ultraviolet light can more effectively excite the fluorescence of GFP than blue light (Figure 23, lower left). Based on his organic chemistry intuition, he inferred that the side chain of serine 65 (Ser65, S65) might be the key to the double peaks. During the discussion, molecular biology expert Geim reminded Professor Qian that he could use site-directed mutagenesis to replace serine 65 with other amino acids to verify this hypothesis. When serine was replaced with threonine (S65T), the ultraviolet excitation peak disappeared, and the fluorescence excitation efficiency of blue light for this GFP was 8 times that of wild-type GFP (Figure 23, lower left and lower right)! Site-directed mutagenesis opened the floodgates for Qian Yongjian's laboratory to comprehensively improve GFP. They successively launched blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), ... adding colorful colors to the "palette" of experimental biologists (Figure 23, lower right).

The 2008 Nobel Prize in Chemistry was eventually shared by Osamu Shimomura, Chalfie and Roger Tsien. Prasher, who had left the academic world for many years, was not favored by the committee due to the number of nominees after experiencing the ups and downs of fate. What is even more regrettable is that the waters near Friday Harbor have been continuously polluted by oil extraction, and the luminous jellyfish have completely disappeared since the mid-1990s.

Recommended Reading

[1] Pieribone, V. & Gruber, DF (2005) Aglow in the Dark: The Revolutionary Science of Biofluorescence, Belknap Harvard.

[2] Shimomura, O. et al (2017) Luminous Pursuit: Jellyfish, GFP, and the Unforeseen Path to the Nobel Prize, World Scientific.

【GFP Discovery History Lecture Video】

Link 1: https://youtu.be/ozjJnNVdzYc

Link 2: https://www.bilibili.com/video/BV17K4y1u7Vv

This article is authorized to be reproduced from the WeChat public account "Medicine Times". The author revised it twice when "Fanpu" was published.

Source: Fanpu