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Chemical Society Reviews www.rsc.org/chemsocrev Volume 38 | Number 10 | October 2009 | Pages 2813–2968 Themed issue: Green fluorescent protein ISSN 0306-0012 Guest editors: Jeremy Sanders and Sophie Jackson TUTORIAL REVIEW HIGHLIGHT Wolf B. Frommer, Michael W. Davidson Marc Zimmer and Robert E. Campbell GFP: from jelly"sh to the Nobel prize Genetically encoded biosensors based and beyond on engineered !uorescent proteins 0306-0012(2009)38:10;1-Q This article was published as part of the 2009 Green Fluorescent Protein issue Reviewing the latest developments in the science of green fluorescent protein Guest Editors Dr Sophie Jackson and Professor Jeremy Sanders All authors contributed to this issue in honour of the 2008 Nobel Prize winners in Chemistry, Professors Osamu Shimomura, Martin Chalfie and Roger Y. Tsien Please take a look at the issue 10 table of contents to access the other reviews HIGHLIGHT www.rsc.org/csr | Chemical Society Reviews GFP: from jellyfish to the Nobel prize and beyondw Marc Zimmer DOI: 10.1039/b904023d On December 10, 2008 Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for ‘‘the discovery and development of the green fluorescent protein, GFP’’. The path taken by this jellyfish protein to become one of the most useful tools in modern science and medicine is described. Osamu Shimomura painstakingly isolated GFP from hundreds of thousands of jellyfish, characterized the chromophore and elucidated the mechanism of Aequorean bioluminescence. Martin Chalfie expressed the protein in E. coli and C. elegans, and Roger Tsien developed a palette of fluorescent proteins that could be used in a myriad of applications. 1. From jellyfish to the as it recognizes the basic research that Aequorea victoria). The jellyfish were Nobel prize Osamu Shimomura did in order to found in the Northeastern Pacific so understand the photophysics involved every summer from 1961 to the eighties ‘‘I decided to find out who the schnook in Aequorean bioluminescence (a field Shimomura and his family would make was that won this year’s prize. So I of research that would probably not be the 5000 km drive from Princeton, opened up my laptop and found out I funded under current funding criteria) New Jersey to the University of was the schnook.’’ That was how Marty and the work of Chalfie and Tsien that Washington’s Friday Harbor laboratory. Chalfie described his discovery that he took an interesting but esoteric protein Jellyfish were abundant and could be had been awarded the Nobel Prize in and made it one of the most useful tools scooped up from a pier using large Chemistry for 2008. He shared the award in modern biology and medicine. It is my shallow nets. Each jellyfish has a couple with Roger Tsien and Osamu Shimomura hope that by the end of this highlight the hundred photoorgans located on the edge ‘‘for the discovery and development of reader will realize that Shimomura, of its umbrella, when stimulated they give the green fluorescent protein, GFP.’’ Chalfie and Tsien are no schnooks and offgreen light, see Fig. 1. This year’s award is particularly interesting that the GFP Nobel award was richly Prior to Shimomura’s jellyfish work, deserved. all known bioluminescent organisms, In August 1960 Osamu Shimomura left such as Cypridina hilgedorfii studied by Chemistry Department, Connecticut College, Japan with a Fulbright Fellowship to Shimomura in Japan2 and the firefly,3 New London, CT06320, USA. E-mail: [email protected] work in the laboratory of Prof. Frank used a luciferin/luciferase system to w Part of a themed issue on the topic of green Johnson at Princeton University. His produce light. Shimomura and Johnson fluorescent protein (GFP) in honour of the project was to elucidate the mechanism discovered that Aequorea victoria was 2008 Nobel Prize winners in Chemistry, of bioluminescence of the jellyfish different. Two proteins were involved in Professors Osamu Shimomura, Martin Chalfie and Roger Y. Tsien. Aequorea aequorea (also known as Aequorea bioluminescence—a calcium binding protein and a green fluorescent protein. In their first summer at Friday Marc Zimmer uses computational methods to examine Harbor Shimomura and Johnson caught the chromophore formation and photophysics of over 10 000 jellyfish from which they fluorescent proteins. He has been a faculty isolated 1 mg of the luminescent calcium member at Connecticut College since 1990. Douglas binding protein which they named Prasher (see this highlight) and Bruce Branchini aequorin.4 In a 1962 paper devoted to (a firefly luciferase chemist at Connecticut College) the extraction, purification and properties introduced him to GFP. Marc wrote Glowing Genes of aequorin,4 the fluorescent protein was the first book to be published about GFP and is described as ‘‘a protein giving solutions responsible for the upkeep of ‘‘The GFP Site’’ at that look slightly greenish in sunlight http://gfp.conncoll.edu. though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite.’’ The green fluorescence Marc Zimmer at the Nobel of the Aequorea light organs had been Award Ceremony described before,5 but this was the first c This journal is The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38,2823–2832 | 2823 During the late seventies Milt Cormier’s laboratory isolated and characterized the proteins involved in the bioluminescence observed in the sea pansy, Renilla.Therearemanysimilarities between Renilla and Aequorea, both have a green fluorescent protein that is excited by radiationless energy transfer from a neighboring blue luminescent protein and both their GFPs have similar but Fig. 1 Aequorea victoria photo organs (left), the whole jellyfish in the dark (middle) and under not identical chromophores.12 Therefore 1 visible light (right). (Photocredits: Steve Haddock and his bioluminescence web page, Monterey it is not surprising that the Cormier Bay Aquarium Research Institute (left image). Osamu Shimomura (right and middle images)). group examined the bioluminescence of both organisms. Isolation, purification time it was shown that the green In 1974 he described the purification and and characterization of these proteins substance responsible for the fluorescence crystallization of GFP, as well as the was painstaking work since thousands was a protein. intermolecular energy transfer between of animals were needed to obtain the Over the next 20 years in order to aequorin and GFP in the jellyfish. This few milligrams required to do the isolate enough of the jellyfish proteins Fo¨ rster-type energy transfer also occurs characterization. Advances in cloning Shimomura caught hundreds of thousands when aequorin and GFP are co-absorbed promised to solve these problems. Bill of jellyfish. They were plentiful at Friday on a Sephadex column, eqn (1).8 Ward, a postdoc in Cormier’s lab, took Harbor ‘‘a constant stream of floating Aequorea GFP and the GFP found in the first step by sequencing Aequorea jellyfish passed along the side of the lab the sea pansy Renilla12 were the only aequorin and GFP, then another dock every morning and evening, riding fluorescent proteins known at the time. postdoc in the lab, Doug Prasher, cloned with the current caused by the tide. One of Shimomura’s most important Aequorea aequorin.18 In Cormier’s lab Sometimes they were extremely abundant, contributions to the field was to determine Prasher also started to clone Aequorea covering the surface of the water.’’6 Once the structure of the chromophore in GFP. He successfully cloned the GFP caught Shimomura used a homemade GFP. He denatured GFP and digested gene from the lab’s Aequorea cDNA jellyfish slicer to cut-offthe part of the it with papain. Only one of the fragments library, but upon sequencing the gene jellyfish umbrella that contained the obtained absorbed above 300 nm and found that it only represented 70% of photoorgans. When the rings of twenty had a similar absorption spectrum to the full-length gene.19 At that time to thirty jellyfish were squeezed through GFP. Although it did not fluoresce Prasher moved from the Cormier lab a rayon gauze, a faintly luminescent it was assumed that this was the and got a position at Woods Hole liquid called squeezate was obtained. In chromophore. Acid hydrolysis, UV and Oceanographic Institute. the squeezate aequorin gives offblue mass spectroscopy as well as synthesis of In the Cormier group the main light upon binding calcium, however in model compounds were used to deter- incentive for the cloning of aequorin the jellyfish radiationless (Fo¨ rster-type) mine a structure for the chromophore and GFP was the production of larger energy transfer occurs and the fluorescent shown in Fig. 2.13 Since then the amounts of the proteins,19 but no one protein absorbs the blue light emitted structure of the chromophore proposed had considered using it as a genetically 14–16 by aequorin (lmax = 470 nm) and fluor- by Shimomura has been confirmed. encoded fluorophore. While at Woods 7,8 esces green (lmax = 509 nm). Hence it Shimomura’s research was basic Hole, Douglas Prasher worked on using was named green fluorescent protein research at its best. He spent more than aequorin as a genetically incorporated (GFP),9 eqn (1). twenty years elucidating the photo- calcium sensor and was the first to get It was easier to isolate aequorin than physics of Aequorea bioluminescence. the idea that GFP could be used in GFP, therefore Shimomura concentrated Shimomura never foresaw the multitude imaging. After collecting more jellyfish most of his research effort on studying of uses for GFP and although he was at Friday Harbor, Prasher sequenced aequorin6,10,11 and he thinks that his best intrigued by the potential uses of aequorin and cloned GFP.20 Fig. 3 lists the DNA work was done in this area, but it is his as a calcium monitor that never drove his 17 research on GFP, the protein associated research.

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