Green Fluorescent Protein | The Embryo Project Encyclopedia (2023)

Green Fluorescent Protein

Green Fluorescent Protein | The Embryo Project Encyclopedia (1)

Editor's note: Anna Guerrero created the above image for this article. You can find the full image and all relevant information here.

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.

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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.

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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.

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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.

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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.

Sources

  1. 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.
  2. Chalfie, Martin. "GFP: Lighting Up Life (Nobel Lecture)." http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ chalfie_lecture.pdf (AccessedFebruary 12, 2014).
  3. Davenport, Demorest and Joseph Nicol. "Luminescence ofHydromedusae." Proceedings of the Royal Society B: BiologicalSciences 144 (1955): 399–11.
  4. 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).
  5. 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).
  6. 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.
  7. Nienhaus, Ulrich. "The Green Fluorescent Protein: A Key Toolto Study Chemical Processes in Living Cells." AngewandteChemie International Edition 47 (2008): 8992–94.
  8. "Osamu Shimomura – Biographical". http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ shimomura-bio.html (AccessedFebruary 12, 2014).
  9. 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.
  10. 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.
  11. 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).
  12. 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).
  13. Tsien, Roger Y. "The Green Fluorescent Protein." AnnualReview of Biochemistry 67 (1998): 509–44.
  14. Tsuji, Frederick. "Early History, Discovery, and Expressionof Aequorea Green Fluorescent Protein, with a Note on anUnfinished Experiment." Microscopy Research and Technique73 (2010): 785–96.
  15. 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.
  16. 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.

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Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia.

Copyright Arizona Board of Regents Licensed as Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported (CC BY-NC-SA 3.0) http://creativecommons.org/licenses/by-nc-sa/3.0/

FAQs

How is green fluorescent protein used in genetic research? ›

Green fluorescent protein (GFP) and its homologs are widely used as fluorescent markers of gene expression and for determination of protein localization and motility in living cells.

What does green fluorescent protein mean? ›

The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP.

How is GFP tagging done? ›

GFP-tagging is a way of preparing a sample for fluorescence microscopy by using the GFP as a fluorescent protein reporter. This is done by cloning the GFP in frame with the target protein at either the N- or C-terminus of the amino acid chain.

How was GFP derived? ›

Green fluorescent protein (GFP) was originally derived from the jellyfish Aequorea victoria (Prendergast and Mann, 1978). It has 238 amino acid residues and a green fluorophore, which is comprised of only three amino acids: Ser65-Tyr66-Gly67.

Can GFP be used in vitro? ›

GFP expression in transduced cells is stable both in vitro and in vivo, and long-term dynamics of GFP-positive fractions in a mixed population can be used to monitor the biological effects of a cotransduced gene.

Is GFP patented? ›

It turns out that sfGFP is patent-protected (lucky we checked!) and that that commercial use requires that a licence fee be paid to the patent-holders.

Why is GFP special? ›

GFP is amazingly useful for studying living cells, and scientists are making it even more useful. They are engineering GFP molecules that fluoresce different colors. Scientists can now make blue fluorescent proteins, and yellow fluorescent proteins, and a host of others.

What is GFP made up of? ›

GFP is a barrel shape with the fluorescent portion (the chromophore) made up of just three amino acids. When this chromophore absorbs blue light, it emits green fluorescence.

Where is GFP found? ›

Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria. The protein is found in the photoorgans of Aequorea, see picture below right.

What is the size of GFP? ›

GFP is a 28 kDa protein that resembles a cylinder with a length of 4.2 nm and a diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel is necessary for its fluorescence and therefore GFP cannot be downsized by deleting residues.

How do you visualize GFP? ›

We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1 microm sections of LR White-embedded material.

What makes GFP glow? ›

Scientists knew that GFP glows because three of its amino acids form a fluorophore, a chemical group that absorbs and emits light.

Who developed GFP? ›

Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, which drifts with the currents off the west coast of North America. He discovered that this protein glowed bright green under ultraviolet light.

When was GFP first discovered? ›

GFP was first discovered fortuitously in 1962 by Shimomura and colleagues during the purification of the bioluminescent protein aequorin from A. victoria.

What is a potential limitation of GFP? ›

However, there are limitations of GFP as a marker (reviewed in [1]). For example, GFP may lose its direct fluorescence during tissue fixation or subsequent processing. For this reason, immunostaining with commercially antibodies is often used for GFP detection.

Is GFP toxic to cells? ›

GFP is cytotoxic by a variety of mechanisms in addition to immunogenicity. Initiation of the apoptosis cascade has been postulated as a possible mechanism for the toxicity of GFP and cellular death.

How do I change the color of my GFP? ›

The simplest way to shift the emission color of GFP is to substitute histidine or tryptophan for the tyrosine in the chromophore, but such blue-shifted point mutants are only dimly fluorescent.

Can you use GFP in vivo? ›

Two-photon absorption of GFP is important for deep-tissue imaging in vivo. Another important feature of fluorescent proteins is the spectral distinction between many members of the family, enabling of multicolor fluorescent proteins to be used simultaneously for multifunctional in vivo imaging.

Can GFP be seen without UV light? ›

It turns out that GFP is amazingly useful in scientific research, because it allows us to look directly into the inner workings of cells. It is easy to find out where GFP is at any given time: you just have to shine ultraviolet light, and any GFP will glow bright green.

Does GFP glow in the dark? ›

Green fluorescent protein (GFP) is a protein that occurs naturally in the jellyfish Aequorea victoria. The purified protein appears yellow under ordinary lighting but glows bright green under sunlight or ultraviolet light.

Can you see GFP with naked eye? ›

The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.

Which applications does GFP have? ›

The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted.

Is GFP an enzyme? ›

The GFP is unique amongst natural pigments for its ability to autocatalyse its own chromophore, requiring only oxygen to complete its synthesis. In this way, a single protein acts as both substrate and enzyme.

How are fluorescent proteins used? ›

Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes.

How can the bioluminescence GFP from jellyfish be used in medical applications? ›

Scientists have developed a process that uses the luminous cells from jellyfish to diagnose cancer tumors deep within the human body. The researchers have used an altered form of the green fluorescent protein (GFP) so that it shows up as red or blue, rather than its original green.

Why does GFP fluoresce green? ›

GFP is a barrel shape with the fluorescent portion (the chromophore) made up of just three amino acids. When this chromophore absorbs blue light, it emits green fluorescence.

Is green fluorescent protein an enzyme? ›

The GFP is unique amongst natural pigments for its ability to autocatalyse its own chromophore, requiring only oxygen to complete its synthesis. In this way, a single protein acts as both substrate and enzyme.

Why do we need fluorescent proteins? ›

Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes.

Why is GFP special? ›

GFP is amazingly useful for studying living cells, and scientists are making it even more useful. They are engineering GFP molecules that fluoresce different colors. Scientists can now make blue fluorescent proteins, and yellow fluorescent proteins, and a host of others.

Where is GFP found? ›

Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria. The protein is found in the photoorgans of Aequorea, see picture below right.

Has GFP been used in humans? ›

Cell Markers: Green Fluorescent Protein (GFP)

GFP and its variants have been used in organisms from bacteria and yeast to mice and human cells.

What gene makes jellyfish glow? ›

The cells that glow turn on the green fluorescent protein (GFP) gene naturally found in jellyfish DNA. The non-glowing cells keep this gene off.

Why do jellyfish glow in the dark? ›

The glow occurs when a substance called luciferin reacts with oxygen. This releases energy, and light is emitted. An enzyme called luciferase facilitates the reaction. Sometimes luciferin and luciferase are bound together with oxygen into a single molecule, or photoprotein.

Can GFP be seen without UV light? ›

It turns out that GFP is amazingly useful in scientific research, because it allows us to look directly into the inner workings of cells. It is easy to find out where GFP is at any given time: you just have to shine ultraviolet light, and any GFP will glow bright green.

Is green fluorescent protein toxic to the living cells? ›

GFP is cytotoxic by a variety of mechanisms in addition to immunogenicity. Initiation of the apoptosis cascade has been postulated as a possible mechanism for the toxicity of GFP and cellular death.

Can you see GFP with naked eye? ›

The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.

What is the size of GFP? ›

GFP is a 28 kDa protein that resembles a cylinder with a length of 4.2 nm and a diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel is necessary for its fluorescence and therefore GFP cannot be downsized by deleting residues.

Which applications does GFP have? ›

The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted.

Who developed GFP? ›

Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, which drifts with the currents off the west coast of North America. He discovered that this protein glowed bright green under ultraviolet light.

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