Green fluorescent protein
- UniProt: P42212
The green fluorescent protein ( GFP abbreviation; . Engl green fluorescent protein) is a first described in 1961 by Osamu Shimomura protein from the jellyfish Aequorea victoria that fluoresces green when excited with blue or ultraviolet light. His invaluable importance in biology, particularly cell biology, is the ability to fuse GFP gene - specific with any other proteins. Due to the fluorescence of GFP can be as the spatial and temporal distribution of the other protein are observed directly in living cells, tissues or organisms.
In 2008, the Nobel Prize in Chemistry for " the discovery and development of green fluorescent protein " Osamu Shimomura to, Martin Chalfie and Roger Tsien were awarded.
Properties
The primary structure consisting of 238 amino acids with a molecular mass of 26.9 kDa. The actual fluorophore of GFP apparently formed autocatalytically from the tripeptide Ser65 - Tyr66 - Gly67 within the polypeptide chain. This fluorescence is not based on a conversion by an external enzyme or subsequently integrated substances, so come completely without any cell-specific processing systems.
In his original organism CFP receives its excitation energy by non-radiative energy transfer from the photoprotein aequorin. In applications CFP is always optically excited. The unmodified, naturally occurring GFP has two excitation maxima. The first is at a wavelength of 395 nm, the second at 475 nm, the emission wavelength is 509 nm
Application
Douglas Prasher isolated and sequenced in 1992, the DNA of GFP. Since it Prasher 1994 managed to use GFP as a marker of other proteins, this technique has become in a few years become a standard method of cell biology. For the production of GFP-fusion proteins, the DNA of the protein of interest to the GFP DNA is linked and placed in a mold (see vector) that can be absorbed by the cell so that they can independently produce the fusion protein (transfection / or transformation in not cell cultures ). In many cases the protein to be assayed is further transported to the correct location in the cell, and the GFP by fluorescence microscopy may be information about the temporal and spatial localization of the target protein in the cell.
GFP is classified in almost all eukaryotic cells as non-toxic and therefore is perfect for the study of biological processes in vivo. The only problem may be the formation of peroxide in the formation of the fluorophore, which could put the cell under stress and damage at very high expression.
Modern methods of fluorescence microscopy, as Vertico SMI, STED microscopy, 3D - SIM microscopy and Photoactivated Localization Microscopy can resolve with GFP derivatives or photoactivatable fluorescent proteins labeled structures on the optical resolution limit.
Also by laboratory methods differing applications, such as a light or a rabbit in the pet trade under the name GloFish available in the U.S., genetically manipulated zebrafish ( Danio rerio) are found.
Variants
Meanwhile, there are various modified versions of the original GFP, which have different fluorescence spectra. According to the color, these are called, for example, CFP (cyan) or YFP (yellow). With skillful application of individual cell organelles are different colorable and means separation (spectral deconvolution ) then observable separately. The crucial point is also the development of enhanced variants such as the enhanced GFP ( eGFP ) or enhanced YFP ( eYFP ) to see.
Ever greater importance are also fluorescent proteins from corals ( Anthozoa ). Foremost among these are the zoanFP ( from Zoanthus sp. ) Or the red fluorescent protein drFP583 ( from Discosoma ), trade name DsRed. Many of these proteins have been mutated and changed in its properties in order to obtain other properties. Many fluorescent proteins are tetramers. This is used by incorporating different monomers rapidly developing. In this case, so the color of the protein changes over time. Such fluorescent timers are useful in order to determine, for example, the age of organelles. The DsRed mutant E5 for example, has this property.
The green fluorescent protein in the art
The German -American artist Julian Voss - Andreae, who specializes in " protein sculptures " in 2004 created a sculpture based on the structure of GFP.