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Green Fluorescent Protein notes.ppt

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Green Fluorescent Protein notes.ppt

  1. 1. Green Fluorescent Protein
  2. 2. GFP Green fluorescent protein (GFP) was originally derived from the jellyfish Aequorea victoria . Aequorea victoria is a jellyfish from which the luminescent protein aequorin and the fluorescent molecule GFP (green fluorescent protein) have been extracted, purified, and eventually cloned. The gene for green fluorescent protein was first cloned in 1992. Since these early studies, GFP has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors that are broadly referred to as fluorescent proteins. These two products have proven useful and popular in various kinds of biomedical research in the 1990s and 2000s and their value is likely to increase in coming years; GFP is particularly easy to use and has wide-ranging value as a fluorescent marker-protein. The luminescent light produced by Aequorea is actually bluish in color, attributable to a molecule known as aequorin, but in a living jellyfish it is emitted via a coupled molecule known as GFP, or green fluorescent protein, which causes the emitted light to appear green to us.
  3. 3. Green fluorescent protein, and its mutated allelic forms, blue, cyan, and yellow fluorescent proteins are used to construct fluorescent chimeric proteins that can be expressed in living cells, tissues, and entire organisms, after transfection with the engineered vectors. Red fluorescent proteins have been isolated from other species, including coral reef organisms, and are similarly useful.
  4. 4. It is not well understood how and why jellyfish use their bioluminescent capabilities, or what biological function this serves. In the laboratory, GFP can be incorporated into a variety of biological systems in order to function as a marker protein. In biological studies, it is extensively used as genetically encoded fluorescent markers. This fluorescent marker enables multicolor labeling and is used in the study of interactions between proteins.
  5. 5. Green fluorescent protein (GFP) It has 238 amino acid residues and a green fluorophore, which is comprised of only three amino acids: Ser65-Tyr66-Gly67. The stable protein structure is formed by beta sheets, which have a conformation that makes up an 11-stranded drum-like structure. The stability of GFP allows it to withstand pH levels ranging from mildly acidic (pH=5.5) to extremely basic (pH=12), and can also resist temperatures of up to 65°C. GFP has major and minor excitation peaks at wavelengths of 395 nm and 475 nm, respectively.
  6. 6. Several modifications have been made from the original GFP, most notably the reduction of the dual excitation peaks of 395 nm and 475 nm down to one excitation peak of 488 nm, which is in the visible blue-light range. The emission peak of original and modified GFP is detected at 509 nm, which is in the visible green region of the electromagnetic spectrum GFP variants with spectra that range from blue to red can be used for live cell– biomaterial interaction imaging.
  7. 7. In order for biomaterial scientists to utilize GFP fusion proteins to capture cellular and subcellular responses to biomaterials, the construction and expression of GFP can be easily accomplished via standard molecular biology techniques. By introducing GFP into host cells, one can visualize GFP-tagged whole-cells, subcellular organisms, and cytoskeletal structure/organization. This form of targeting allows microscopist and biomaterial scientists alike to study cellular behavior by observing GFP-tagged proteins and capture information at a level that was previously inaccessible, both spatially and temporally
  8. 8. Primary & Secondary Structure A 21 kDa protein consisting of 238 residues strung together to form a secondary structure of five α-helices and one eleven-stranded β-pleated sheet, where each strand contains nine to thirteen residues each. 11-strand β-barrel, with an α-helical segment threaded up through the interior of the barrel Structurally, the barrel α-helical segment is near the center of the β-barrel cavity.
  9. 9. The Flourophore GFP is a β-barrel protein. Within an hour or so after synthesis and folding, a self-catalyzed maturation process occurs in the protein, whereby adjacent serine, glycine, and tyrosine side chains in the interior of the barrel react with each other and with oxygen to form a fluorophore covalently attached to a through-barrel α-helical segment, near the center of the β-barrel cavity. The GFP fluorophore thus produced is excited by the absorption of blue light from the fluorescence microscope, and then decays with the release of green fluorescence.
  10. 10. Using GFP as a Research Tool The Green fluorescent proteins (GFPs) has been successfully used as a reporter gene to detect gene expression in a variety of cells. GFP is widely used to probe the events that occur within living cells , including protein localization in eukaryotic cells . It has several advantages over other reporter molecules: It has been used as a reporter for protein localization in Escherichia coli . GFP has several features that make it an attractive candidate for protein localization studies in bacteria. For example, the protein is active in E. coli, and it has proven to be a useful reporter for a number of investigations in this microorganism, including monitoring gene expression , assessing viability , and detecting bacteria in the environment . GFP is active as a chimeric protein and has provided details of bacterial cell division and chromosome partitioning.
  11. 11. An additional characteristic of GFP that is potentially advantageous for protein localization studies is that GFP has a small molecular mass of 27 kDa. The three-dimensional structure of GFP is also known and reveals that GFP attains a relatively uncomplicated “β-can” structure. This fact suggests that GFP would likely be exported from the cytoplasm if fused to appropriate export signals. Also, GFP emits green light following excitation of an internal fluorophore composed of a Ser- Tyr-Gly sequence positioned near the protein's amino terminus. Excitation of GFP-expressing cells can be performed by exposure to long-wave UV light, making detection of GFP activity simple and obviating the need for specific substrates
  12. 12. Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. the genes that encode chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), firefly luciferase , and Renilla luciferase have been used extensively as quantitative reporters of viral and cellular promoter activity. However, these conventional reporter genes share two disadvantages. First, most reporter assays provide a measure of average promoter activity over the entire cell population sampled, but provide no measure of the variation in promoter activity that exists between individual cells in the population. The second limitation of conventional reporter genes is that kinetic analysis of promoter activity quickly becomes unmanageable because there is no way to monitor gene expression over time
  13. 13. Since the cloning and enhancement of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria, GFP has been widely used as a reporter gene. In particular, GFP has been used extensively to visualize spatial and temporal patterns of gene expression in vivo and to study intracellular patterns of protein localization and trafficking. Several studies suggest that it is possible that GFP may also be used as a quantitative reporter of gene expression in eukaryotic cells A major advantage of GFP is that intracellular accumulation of the protein can be directly observed in living cells over time. Digital photographs of GFP-expressing cells provide a reasonable approximation of the quantitative results. GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Informative information analysed in the Publication below: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1242169/
  14. 14. Answer the question below Based on your reading and understanding of GFP, DISCUSS ITS ADVANTAGES OVER OTHER REPORTER GENES to detect gene expression in a variety of cells either in prokaryotic or eukaryotic cells.
  15. 15. Using GFP as a Research Tool 1. Fluorescence resonance energy transfer (FRET). 2. Microscopy: Fluorescence microscopy Laser scanning confocal fluorescence microscopy Other tools include Centrifugation: density gradient and rate zonal centrifugation Electroporesis: SDS-PAGE
  16. 16. A. Optical layout of a fluorescence microscope. Incident light tuned to excite the fluorescent molecule is reflected by a dichroic mirror, and then focused on the sample; fluorescent light (longer wavelength than excitation light) emitted by the sample passes through the dichroic mirror for viewing. B. Immunofluorescent micrograph of a human skin fibroblast, stained with fluorescent antiactin antibody. Cells were fixed, permeabilized, and then incubated with fluorescein-coupled antibody. Unbound antibody was washed away before viewing FLUORESCENCE MICROSCOPE

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