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Green fluorescent protein (GFP) is a protein in thejellyfish AequoreaVictoria that exhibits green fluorescence when exposed tolight. The protein has 238 amino acids, three of them (Numbers 65 to 67)form a structure that emits visible green fluorescent light. Inthe jellyfish, GFP interacts with another protein, called aequorin,which emits blue light when added with calcium. Biologists use GFPto study cells in embryos and fetuses during developmentalprocesses.
Biologists use GFP as a marker protein. GFP can attach to andmark another protein with fluorescence, enabling scientists to seethe presence of the particular protein in an organic structure.Gfp refers to the gene that produces green fluorescentprotein. Using DNA recombinant technology, scientists combine theGfp gene to a another gene that produces a protein that they want to study,and then they insert the complex into a cell. If the cell producesthe green fluorescence, scientists infer that the cell expresses thetarget gene as well. Moreover, scientists use GFP to label specificorganelles, cells, tissues. As the Gfp gene is heritable, thedescendants of labeled entities also exhibit green fluorescence.
Edmund N. Harvey, a professor at Princeton University inPrinceton, New Jersey, initiated the studies on bioluminescence inthe US. In 1921, Harvey described the yellow tissues in the umbrellaof jellyfish as being luminous in particular conditions, such as atnight or when the jellyfish is stimulated with electricity. In 1955,Demorest Davenport at the University of California at Santa Barbarain Santa Barbara, California, and Joseph Nicol at Plymouth MarineLaboratory in Plymouth, England, used photoelectric recording andhistological methods to confirm Harvey's descriptions, and theyidentified the green fluorescent materials in the marginal canal ofthe umbrella.
In the same year, Osamu Shimomura became a research assistant atNagoya University in Nagoya, Japan, and he crystallized the luciferin,a light-emitting compound found in the sea-firefly Vargulahilgendorfii. Shimomura published his results in 1957. Oneof Harvey's students, Frank H. Johnson, studied bioluminescence atPrinceton University. Johnson followed Shimomura's work and invitedhim to work in the US, and in 1960 Shimomura received aFulbright Travel Grant and started working with Johnson. Shortlyafter Shimomura arrived in the US, Johnson introduced thebioluminescence of Aequorea Victoria to Shimomura. In the US,jellyfish live only on the west coast, so Shimomura traveled to theFriday Harbor Laboratories of the University of Washington in SanJuan Island, Washington, during the summer of 1961. After catchingabout 10,000 jellyfish, Shimomura took the extracts of the jellyfishand preserved it in dry-ice to bring it back to Princeton inSeptember of 1961.
At Princeton, Shimomura and his colleagues started to purify thebioluminescent substance, and they found that it was a protein,which they called aequorin. When they purified aequorin, they alsodiscovered traces of another protein, which showed greenfluorescence. Shimomura's team published the findings in "Exraction,Purification, and Properties of Aequorin" in 1962. The paper wasabout aequorin, but it also described a green protein, whichexhibited green fluorescence under sunlight. John W. Hasting andJames G. Morin, who later researched aequorin, termed the proteinas green fluorescent protein in 1971.
Shimomura focused on aequorin, purified the protein, crystallizedit, and elucidated its underlying structure. He also studied theproperties of GFP, and published his last paper on GFP in 1979. In1981, after leaving Princeton University for the Marine BiologyLaboratory in Woods Hole, Massachusetts, Shimomura did not research on GFP anymore. From 1979 to 1992, many researchersstudied various aspects of GFP, including the use of NuclearMagnetic Resonance to study the amino acids of the protein, the useof X-rays to study its crystal, and the evolution of GFP.
In the early 1990s, molecular biologist Douglas Prasher,at the Marine Biology Laboratory, used GFP to design probes, atechnology involving fragments of DNA to detect the presence ofnucleotide sequences. Prasher isolated the complementary DNA (cDNA)of Gfp gene, and he published the sequence of the gene in 1992.After the publication of the cDNA sequence in 1992, Prasher's funding from theAmerican Cancer Society in Atlanta, Georgia, expired. When he appliedfor funding from the US National Institute of Health in Bethesda,Maryland, the reviewer argued that Prasher's research lackedcontributions to society. As Prasher could not secure funding tosupport his research any further, he left the Marine BiologyLaboratory to work for the US Department of Agriculture inMassachusetts.
After Prasher's publication in 1992, many scientists tried totransfer and express the Gfp gene in organisms other thanjellyfish using DNA recombinant technology, and Martin Chalfie wasthe first who succeeded. Chalfie, a Professor at Columbia Universityin New York, New York, studied the development of the nematode Caenorhabditiselegans. Chalfie heard about the protein GFP in a lecture,and he speculated that GFP might facilitate his study of geneexpression in C. elegans. Chalfie's team obtained the cDNA ofthe gene Gfp from Prasher and inserted only the codingsequence of Gfp gene first in the bacterium EscherichiaColi, and then in C. elegans. Chalfie and his team foundthat Gfp gene produced GFP without added enzymes orsubstrates in both organisms. In 1994, Chalfie published his resultsin "Green Fluorescent Protein as a Marker for Gene Expression". Thedetection of GFP needed only ultraviolet light. Thereafter, manybiologists introduced GFP into their experiments to study geneexpression. Satoshi Inouye and Frederick Tsuji at PrincetonUniversity also expressed Gfp in E. Coli in 1994.
Many scientists tried to mutate the Gfp gene to make the resultant proteinreact to wider wavelengths and emanate different colors. Otherscientists studied different fluorescent proteins (FPs). RogerTsien, a professor at the University of California San Diego, in SanDiego, California, reengineered the gene Gfp to produce theprotein in different structures. His team also reengineered other FPs.Due to Tsien's and other bioengineers' efforts, GFP could not onlyexhibit brighter fluorescence, but also respond to a wider range ofwavelengths, as well as emit almost all colors, except for red.Tsien's findings enabled scientists to tag multiple colored GFPs todifferent proteins, cells, or organelles of interest, and scientistscould study the interaction of those particles. Red FP becameavailable in 1999, when Sergey Lukyanov's team at theShemyakin-Ovchinnikov Institute of Bioorganic Chemistry in Moscow,Russia, found that some corals contained the red fluorescentprotein, called DsRed. Other laboratories developed fluorescentsensors for calcium, protease and other biological molecules. Sincethen, scientists have reported more than 150 distinct GFP-likeproteins in many species.
As GFP does not interfere with biological processes when usedin vivo, biologists use it to study how organisms develop.For example, after 1994, Chalfie and his colleagues applied GFP inthe study of the neuron development of C. elegans. In a 2002paper, Chalfie and his colleagues describe how they first labeled aspecific gene involved in tactile perception in neuron cells withGFP, and then observed the amount of fluorescence emitted by thosecells. Because mutant cells produced less or more GFP than normalcells, the abnormal amount of fluorescence production indicated theabnormal development of mutants. Since then, this field of researchexpanded to many other organisms, including fruitflies, mice, andzebra fish.
On 10 December 2008, The Royal Swedish Academy of Science academyawarded the Noble Prize in Chemistry to Tsien, Chalfie, andShimomura for their discoveries on GFP.
- Chalfie, Martin,Yuan Tu, Ghia Euskirchen, William W. Ward, and Douglas C.Prasher. "Green Fluorescent Protein as a Marker for GeneExpression." Science 263 (1994): 802–05.
- Chalfie, Martin. "GFP: Lighting Up Life (Nobel Lecture)." http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ chalfie_lecture.pdf (AccessedFebruary 12, 2014).
- Davenport, Demorest and Joseph Nicol. "Luminescence ofHydromedusae." Proceedings of the Royal Society B: BiologicalSciences 144 (1955): 399–11.
- Harvey, Edmund. "Studies on bioluminescence. XIII.Luminescence in the coelenterates." Biological Bulletin41 (1921): 280–87. http://archive.org/details/jstor-1536528 (Accessed February 21, 2014).
- Hastings, John, and James Morin. "Comparative Biochemistryof Calcium Activated Photo Proteins from the Ctenophore,Mnemiopsis and the Coelenterates Aequorea, Obelia, Pelagia andRenilla." The Biological Bulletin 137 (1969): 402. http://www.biolbull.org/content/137/2/384.full.pdf (AccessedFebruary 11, 2014).
- Matz, Mikhail V., Arkady F. Fradkov, Yulii A. Labas,Aleksandr P. Savitsky, Andrey G. Zaraisky, Mikhail L. Markelov,and Sergey A. Lukyanov. "Fluorescent proteins fromnonbioluminescent Anthozoa species." Nature Biotechnology17 (1999): 969–73.
- Nienhaus, Ulrich. "The Green Fluorescent Protein: A Key Toolto Study Chemical Processes in Living Cells." AngewandteChemie International Edition 47 (2008): 8992–94.
- "Osamu Shimomura – Biographical". http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ shimomura-bio.html (AccessedFebruary 12, 2014).
- Prasher, Douglas C, Virgina K. Eckenrodeb, William W. Ward,Frank G. Prendergastd, and Milton J. Cormier. "Primary Structureof the Aequorea Victoria Green-Fluorescent Protein." Gene111 (1992): 229–33.
- Shimomura, Osamu, Frank H. Johnson, and Yo Saiga."Extraction, Purification and Properties of Aequorin, aBioluminescent Protein from Luminous Hydromedusan Aequorea."Journal of Cellular and Comparative Physiology 59 (1962):223–39.
- Shimomura, Osamu. "Structure of the Chromophore of AequoreaGreen Fluorescent Protein." FEBS Letter 104 (1979):220–22. http://dx.doi.org/10.1016/0014-5793(79)80818-2 (Accessed February 21,2014).
- Shimomura, Osamu. "The Discovery of Aequorin and GreenFluorescent Protein." Journal of Microscopy 217 (2005):3–15. "The Nobel Prize in Chemistry 2008". http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ (Accessed February 12, 2014).
- Tsien, Roger Y. "The Green Fluorescent Protein." AnnualReview of Biochemistry 67 (1998): 509–44.
- Tsuji, Frederick. "Early History, Discovery, and Expressionof Aequorea Green Fluorescent Protein, with a Note on anUnfinished Experiment." Microscopy Research and Technique73 (2010): 785–96.
- Zhang, Yun, Charles Ma, Thomas Delohery, Brian Nasipak,Barrett C. Foat, Alexander Bounoutas, Harmen J. Bussemaker,Stuart K. Kim, and Martin Chalfie. "Identification of genesexpressed in C. elegans touch receptor neurons." Nature418 (2002): 331–35.
- Zimmer, Mark. "GFP: from Jellyfish to the Nobel Prize andBeyond." Chemical Society Reviews 38 (2009): 2823–32. http://pubs.rsc.org/en/content/articlepdf/2009/cs/b904023d(Accessed February 12, 2014).
Zou, Yawen, "Green Fluorescent Protein". Embryo Project Encyclopedia (2014-06-11). ISSN: 1940-5030 http://embryo.asu.edu/handle/10776/7903.
Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia.
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