Download GREEN FLUORESCENT PROTEIN TAGGING IN MOLECULAR

GREEN FLUORESCENT PROTEIN TAGGING IN MOLECULAR BIOLOGY
GFP revolutionized molecular biology because it allows scientists to see structures that could only
imagine earlier. By attaching GFP to other cell proteins, researchers can easily visualize movement and
therefore function of biological systems.
GFP STRUCTURE:
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9kDa) that
exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Although many
other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein
first isolated from the jellyfish Aequorea victoria.
Eleven beta-strands make up the beta-barrel and an alpha-helix runs through the center. The
chromophore is located in the middle of the beta-barrel, it is occasionally referred to as the “light in the
can.”
Among the most important aspects of the green fluorescent protein to appreciate is that the entire 27
kiloDalton native peptide structure is essential to the development and maintenance of its fluorescence.
It is remarkable that the principle fluorophore is derived from a triplet of adjacent amino acids: the
serine, tyrosine, and glycine residues at locations 65, 66, and 67 (referred to as Ser65, Tyr66, and Gly67).
Although this simple amino acid motif is commonly found throughout nature, it does not generally result
in fluorescence. What is unique to the fluorescent protein is that the location of this peptide triplet
resides in the center of a remarkably stable barrel structure consisting of 11 beta-sheets folded into a
tube. This tight protein structure also confers resistance to fluorescence variations due to fluctuations in
pH, temperature, and denaturants such as urea.
GFP HISTORY:
The gene for green fluorescent protein was first cloned in 1992, the significant potential as a molecular
probe was not realized until several years later when fusion products were used to track gene
expression in bacteria and nematodes. Since these early studies, green fluorescent protein has been
engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors that
are broadly referred to as fluorescent proteins. More recently, fluorescent proteins from other species
have been identified and isolated, resulting in further expansion of the color palette.
USES:
Fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has
triggered the development of highly automated live-cell fluorescence microscopy systems, which can be
used to observe cells over time expressing one or more proteins tagged with fluorescent proteins. For
example, GFP had been widely used in labelling the spermatozoa of various organisms for identification
purposes as in Drosophila melanogaster, where expression of GFP can be used as a marker for a
particular characteristic. GFP can also be expressed in different structures enabling morphological
distinction.
Another powerful use of GFP is to express the protein in small sets of specific cells. This
allows researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the
living organism). Genetically combining several spectral variants of GFP is a useful trick for the analysis
of brain circuitry (Brainbow - see below).
Other interesting uses of fluorescent proteins in the
literature include using FPs as sensors of neuron membrane potential, tracking of AMPA receptors on
cell membranes, viral entry and the infection of individual influenza viruses and lentiviral viruses.
As an example, it has also been found that new lines of transgenic GFP rats can be relevant for gene
therapy as well as regenerative medicine. By using "high-expresser" GFP, transgenic rats display high
expression in most tissues, and many cells that have not been characterized or have been only poorly
characterized in previous GFP-transgenic rats.
Time lapse movies has redefined the understanding of many biological processes including protein
folding, protein transport, and RNA dynamics
GFP MUTANTS
In order to adapt fluorescent proteins for use in mammalian systems, several basic modifications of the
wild-type green fluorescent protein were undertaken and are now found in all commonly used variants.
The first step in 1995 was to optimize the maturation of fluorescence to a 37-degree Celsius
environment. Maturation of the wild-type fluorophore is quite efficient at 28 degrees, but increasing the
temperature to 37 degrees substantially reduces overall maturation and results in decreased
fluorescence. In addition to improving the maturation at 37 degrees, the optimization of codon usage
for mammalian expression has also improved overall brightness of green fluorescent protein expressed
in mammalian cells. In all, over 190 silent mutations have been introduced into the coding sequence to
enhance expression in human tissues including Superfolder GFP, a series of mutations that allow GFP to
rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.
In the Brainbow mice, the Harvard researchers have introduced genetic machinery that randomly mixes
green, cyan and yellow fluorescent proteins in individual neurons thereby creating a palette of ninety
distinctive hues and colors. "The technique drives the cell to switch on fluorescent protein genes in
neurons more or less at random
pH-sensitive mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid
change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to
visualize synaptic activity in neurons.
Here's a video visualization of mitosis using GFP.
http://www.youtube.com/watch?v=nR64M7GWiWc
SPLIT GFPs:
Split GFPs are proteins that have been separated into two polypeptides that together compose the
entire primary sequence of GFP. Many split GFPs that do not spontaneously reassemble into a GFP-like
structure can be reassembled by fusing both halves of GFP to interacting proteins. Because the split
GFPs require fused interacting proteins to form the chromophore, the primary use of split GFPs is for
imaging protein-protein interactions in vivo. Although split GFPs are widely used for imaging in vivo,
little is known about the reassembly mechanism.
HOW GFP IS SPLICED
GFP is attached to a protein of interest by inserting the GFP gene between the regulatory sequence (e.g.
promotor) and gene of interest; that is, the gene's regulatory sequence now controls the production of
GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins
are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is
expressed, or the target proteins are produced, will fluoresce when observed under fluorescence
microscopy. Through its ability to form internal chromophore without requiring accessory cofactors,
enzymes or substrates other than molecular oxygen, GFP makes for an excellent tool in all forms of
biology.