AAPI in the discovery and engineering of green fluorescent protein

The green fluorescent protein (GFP), and its derivatives, remain the most important discoveries in the fields of cellular and molecular biology and biotechnology. Three AAPI scientists, Dr. Osamu Shimomura, Dr. Roger Tsien, and Dr. Frederick Tsuji, pioneered the extraction and bioengineering as well as characterized the properties of this incredible glowing protein. Tsien and Shimomura were awarded the Nobel Prize in Chemistry with Dr. Martin Chalfie in 2008.

In the 1960s, along with colleagues Dr. Frank H. Johnson and Yo Saiga at Princeton University, Osamu Shimomura first isolated wild-type GFP from Aequorea victoria, a jellyfish species sampled from the west coast of San Juan Island, Washington. Dr. Robert Fernald, of the University of Washington Friday Harbor Laboratories hosted their residences. Shimomura, Johnson, and Saiga found that under ultraviolet light, the extracted protein glowed brightly. Shimomura later took a faculty position at the Woods Hole Marine Biological Laboratory and continued his studies on marine bioluminescence.

Frederick Tsuji and his colleague Satoshi Inouye at Scripps Institution of Oceanography, University of California, San Diego (UCSD), were among the first scientists to express GFP in a heterologous system through genetic manipulation. From the 1960s to the late 1980s, though the green fluorescent protein had been discovered, it was not yet adopted as a tool for molecular biology. In 1992, Dr. Douglas Prasher at Woods Hole Oceanographic Institution reported the nucleotide sequence of the wildtype GFP (Side story: Dr. Prasher had to leave science due to grants being turned down. He then became a bus driver. When GFP was awarded the Nobel Prize of Chemistry in 2008, his contribution was significantly recognized as essential by the two laureates Dr. Martin Chalfie and Dr. Roger Tsien. He then finally got the chance to return to science, working in the Tsien lab at UCSD). Dr. Prasher generously shared his findings and the cDNA with several labs, including Tsuji’s lab. Tsuji and Inouye expressed GFP in E. coli cells, which independently repeated the success one month after Chalfie’s successful expression of GFP in C. elegans and E. coli.

Roger Y. Tsien, with his colleagues Roger Heim and Andrew B. Cubitt at the Howard Hughes Medical Institute and University of California, San Diego, resolved the underlying mechanisms of how GFP fluoresces. They introduced mutations to the protein sequence and successfully improved the performance and stability of the fluorescence. They also extended the fluorescing colors from green to a broader spectrum of different colors. This enables researchers to follow different biological pathways in parallel in the same cell via different tagging colors .

Today, these three great stars are gradually fading away from us. Shimomura passed away in 2018. Tsuji passed away in 2016, as did Tsien. At UCSD, Only the FedEx delivery person remembers where the Tsien lab is, who Roger Tsien was and no one knows where Douglas Prasher is now after the Tsien lab dissolved. However, their legacy has been engraved into the human history of scientific exploration. GFP illuminated the world of molecular biology research. Its nontoxicity, inheritability and non-interference with other biological processes make it a great in-vivo and intracellular biomarker, benefiting a broad range of biological research.

Brief Biography

Frederick Ichiro Tsuji was born on August 23, 1923 in Honolulu. He got his bachelors, masters and Ph.D. degrees from Cornell University. His professional career started as an Assistant professor at Duquesne University, Pittsburgh. He then served as a research assistant at Princeton University from 1952 to 1955. In 1972, he moved to the west coast and became a research professor at USC. He then directed the NSF biochemistry program from 1976--1978. At last, he settled down at La Jolla and became a research biochemist and professor at Scripps Institute of Oceanography, UCSD.

Osamu Shimomura (下村 脩) was born on August 27, 1928, in Fukuchiyama, Japan. His high school graduation coincided with the end of World War 2, which posed many challenges for him entering college. He finally got into Nagasaki Pharmacy College, where he built up his interest in chemical experiments. He did chemical extractions and chromatography experiments under the supervision of Professor Shungo Yasunaga. Afterwards, he joined the Yoshimasa Hirata laboratory at Nagoya University as a research student (only for study, not pursuing any degree) in 1955. At Nagoya, he accepted the huge challenge of extracting and crystallizing Cypridina luciferin, a task that was riddled with uncertainties and many previously failed attempts. Shimomura tackled the challenge in 1957. In 1959, he got a job offer from Dr. Frank Johnson of Princeton. Dr. Hirata kindly awarded Shimomura a doctorate degree though it was not his initial intention. The unexpected doctorate degree benefited Shimomura with a higher salary at Princeton. And in 1961, with Dr. Johnson, Shimomura conducted the famous GFP extraction experiment. He went back to Nagoya University as an associate professor in 1963. In early 1965, he went to New Zealand to study different bioluminescence organisms. And in late 1965, he returned to Princeton with his family and stayed there until 1978. His research at Princeton was focused on the bioluminescence of different marine organisms and the protein structure of Aequorin. In 1981, he moved to the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, as a tenured senior scientist.

Roger Yonchien Tsien (錢永健) was born on February 1, 1952 in New York. His ancestors were the “scholar-gentry” class in Hangzhou, China, where Tsien (or more commonly today as Qian) is a common family name. He is the nephew of Xuesen Qian, the co-founder of Jet Propulsion Laboratory (JPL) and the founder of engineering cybernetics. His father, Hsue-Chu Tsien was an aeronautic engineer who graduated from MIT. At the age of 8, Tsien became obsessed with the pretty colors in chemistry when he was carrying out experiments of the reactions between metal salts and silicate or cellulose. Similar to many children of immigrants, and contrary to interest in science, he felt that his mother’s Chinese language lessons were tedious. He was sponsored by the National Science Foundation for a summer research program at Ohio University in 1967. Under the supervision of Dr. Robert Kline, he conducted experiments to test out how metals bind to thiocyanate. The research was premature and did not yield any sound conclusions (Tsien’s personal assessment), and Tsien had no idea why he got the first prize in the Westinghouse science talent search, remarking “I retain a dislike for scientific competitions,”. In 1968, he attended Harvard University, where he gradually lost interest in chemistry and got into the field of molecular biology and neurobiology. After earning his bachelors degree in 1972, he moved from Cambridge, MA to Cambridge, UK, and conducted his graduate research under the supervision of Richard Hume Adrian. From 1982 to 1989, He was appointed as an assistant professor at the University of California, Berkeley. Beginning in 1989, he worked at the University of California, San Diego, as Professor of Pharmacology and Professor of Chemistry and Biochemistry and as an investigator of the Howard Hughes Medical Institute.