As an example, a single point mutation to the native jellyfish peptide creates a photoactivatable version of green fluorescent protein known as PA-GFP that enables photoconversion of the excitation peak from ultraviolet to blue by illumination with light in the nanometer range. Unconverted PA-GFP has an excitation peak similar in profile to that of the wild type protein approximately to nanometers. After photoconversion, the excitation peak at nanometers increases approximately fold.
This event evokes very high contrast differences between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of molecular subpopulations within a cell. Illustrated in Figure 6 a is a transfected living mammalian cell containing PA-GFP in the cytoplasm being imaged with nanometer argon-ion laser excitation before Figure 6 a and after Figure 6 d photoconversion with a nanometer blue diode laser.
Other fluorescent proteins can also be employed as optical highlighters. Three-photon excitation at less than nanometers of DsRed fluorescent protein is capable of converting the normally red fluorescence to green. This effect is likely due to selective photobleaching of the red chromophores in DsRed, resulting in observable fluorescence from the green state. The Timer variant of DsRed gradually turns from bright green nanometer emission to bright red nanometer emission over the course of several hours.
The relative ratio of green to red fluorescence can then be used to gather temporal data for gene expression investigations. A photoswitchable optical highlighter, termed PS-CFP , derived by mutagenesis of a green fluorescent protein variant, has been observed to transition from cyan to green fluorescence upon illumination at nanometers note photoconversion of the central cell in Figures 6 b and 6 e.
Expressed as a monomer, this probe is potentially useful in photobleaching, photoconversion and photoactivation investigations. Additional mutagenesis of this or related fluorescent proteins has the potential to yield more useful variants in this wavelength region. Optical highlighters have also been developed in fluorescent proteins cloned from coral and anemone species. Kaede , a fluorescent protein isolated from stony coral, photoconverts from green to red in the presence of ultraviolet light.
Unlike PA-GFP, the conversion of fluorescence in Kaede occurs by absorption of light that is spectrally distinct from its illumination. Unfortunately, this protein is an obligate tetramer, making it less suitable fur use as an epitope tag than PA-GFP. Another tetrameric stony coral Lobophyllia hemprichii fluorescent protein variant, termed EosFP see Table 2 , emits bright green fluorescence that changes to orange-red when illuminated with ultraviolet light at approximately nanometers.
In this case, the spectral shift is produced by a photo-induced modification involving a break in the peptide backbone adjacent to the chromophore. Further mutagenesis of the "wild type" EosFP protein yielded monomeric derivatives, which may be useful in constructing fusion proteins. A third non- Aequorea optical highlighter, the Kindling fluorescent protein KFP1 has been developed from a non-fluorescent chromoprotein isolated in Anemonia sulcata , and is now commercially available Evrogen.
Kindling fluorescent protein does not exhibit emission until illuminated with green light. Low-intensity light results in a transient red fluorescence that decays over a few minutes see the mitochondria in Figure 6 c.
Illumination with blue light quenches the kindled fluorescence immediately, allowing tight control over fluorescent labeling. In contrast, high-intensity illumination results in irreversible kindling and allows for stable highlighting similar to PA-GFP Figure 6 f.
The ability to precisely control fluorescence is particular useful when tracking particle movement in a crowded environment. For example, this approach has been successfully used to track the fate of neural plate cells in developing Xenopus embryos and the movement of individual mitochondria in PC12 cells. As the development of optical highlighters continues, fluorescent proteins useful for optical marking should evolve towards brighter, monomeric variants that can be easily photoconverted and display a wide spectrum of emission colors.
Coupled with these advances, microscopes equipped to smoothly orchestrate between illumination modes for fluorescence observation and regional marking will become commonplace in cell biology laboratories. Ultimately, these innovations have the potential to make significant achievements in the spatial and temporal dynamics of signal transduction systems.
Fluorescent proteins are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These ubiquitous probes have been extremely useful as reporters for gene expression studies in cultured cells and tissues, as well as living animals.
In live cells, fluorescent proteins are most commonly employed to track the localization and dynamics of proteins, organelles, and other cellular compartments. A variety of techniques have been developed to construct fluorescent protein fusion products and enhance their expression in mammalian and other systems.
The primary vehicles for introducing fluorescent protein chimeric gene sequences into cells are genetically engineered bacterial plasmids and viral vectors.
Fluorescent protein gene fusion products can be introduced into mammalian and other cells using the appropriate vector usually a plasmid or virus either transiently or stably.
In transient, or temporary, gene transfer experiments often referred to as transient transfection , plasmid or viral DNA introduced into the host organism does not necessarily integrate into the chromosomes, but can be expressed in the cytoplasm for a short period of time. Expression of gene fusion products, easily monitored by the observation of fluorescence emission using a filter set compatible with the fluorescent protein, usually takes place over a period of several hours after transfection and continues for 72 to 96 hours after introduction of plasmid DNA into mammalian cells.
In many cases, the plasmid DNA can be incorporated into the genome in a permanent state to form stably transformed cell lines.
The choice of transient or stable transfection depends upon the target objectives of the investigation. The basic plasmid vector configuration useful in fluorescent protein gene transfer experiments has several requisite components. The plasmid must contain prokaryotic nucleotide sequences coding for a bacterial replication origin for DNA and an antibiotic resistance gene.
These elements, often termed shuttle sequences, allow propagation and selection of the plasmid within a bacterial host to generate sufficient quantities of the vector for mammalian transfections.
In addition, the plasmid must contain one or more eukaryotic genetic elements that control the initiation of messenger RNA transcription, a mammalian polyadenylation signal, an intron optional , and a gene for co-selection in mammalian cells.
Transcription elements are necessary for the mammalian host to express the gene fusion product of interest, and the selection gene is usually an antibiotic that bestows resistance to cells containing the plasmid. These general features vary according to plasmid design, and many vectors have a wide spectrum of additional components suited for particular applications. Illustrated in Figure 7 is the restriction enzyme and genetic map of a commercially available BD Biosciences Clontech bacterial plasmid derivative containing the coding sequence for enhanced yellow fluorescent protein fused to the endoplasmic reticulum targeting sequence of calreticulin a resident protein.
Expression of this gene product in susceptible mammalian cells yields a chimeric peptide containing EYFP localized to the endoplasmic reticulum membrane network, designed specifically for fluorescent labeling of this organelle.
The host vector is a derivative of the pUC high copy number approximately plasmid containing the bacterial replication origin, which makes it suitable for reproduction in specialized E.
The kanamycin antibiotic gene is readily expressed in bacteria and confers resistance to serve as a selectable marker. Additional features of the EYFP vector presented above are a human cytomegalovirus CMV promoter to drive gene expression in transfected human and other mammalian cell lines, and an f1 bacteriophage replication origin for single-stranded DNA production. The vector backbone also contains a simian virus 40 SV40 replication origin, which is active in mammalian cells that express the SV40 T-antigen.
Six unique restriction enzyme sites see Figure 7 are present on the plasmid backbone, which increases the versatility of this plasmid. Successful mammalian transfection experiments rely on the use of high quality plasmid or viral DNA vectors that are relatively free of bacterial endotoxins.
In the native state, circular plasmid DNA molecules exhibit a tertiary supercoiled conformation that twists the double helix around itself several times.
For many years, the method of choice for supercoiled plasmid and virus DNA purification was cesium chloride density gradient centrifugation in the presence of an intercalation agent such as ethidium bromide or propidium iodide. This technique, which is expensive in terms of both equipment and materials, segregates the supercoiled plasmid DNA from linear chromosomal and nicked circular DNA according to buoyant density, enabling the collection of high purity plasmid DNA.
Recently, simplified ion-exchange column chromatography methods commonly termed a mini-prep have largely supplanted the cumbersome and time-consuming centrifugation protocol to yield large quantities of endotoxin-free plasmid DNA in a relatively short period of time.
Specialized bacterial mutants, termed competent cells, have been developed for convenient and relatively cheap amplification of plasmid vectors.
The bacteria contain a palette of mutations that render them particularly susceptible to plasmid replication, and have been chemically permeabilized for transfer of the DNA across the membrane and cell wall in a procedure known as transformation. After transformation, the bacteria are grown to logarithmic phase in the presence of the selection antibiotic dictated by the plasmid.
The bacterial culture is concentrated by centrifugation and disrupted by lysis with an alkaline detergent solution containing enzymes to degrade contaminating RNA. The lysate is then filtered and placed on the ion-exchange column. Alcohol isopropanol precipitation concentrates the eluted plasmid DNA, which is collected by centrifugation, washed, and redissolved in buffer. Endogenous proteins do not contain protein or peptide tags and therefore are sometimes difficult to detect in an assay.
One solution that enables easy detection is to genetically fuse protein and peptide tags to the protein of interest. Tagged proteins can be used for purposes such as immunoprecipitation, microscopy, protein purification, Western blotting, protein arrays, etc. GFP-tagged proteins are often used for fluorescence microscopy, immunoprecipitation, protein purification, and Western blotting. GFP from A.
Its emission peak is at nm. Many fluorescent proteins are based on the GFP sequence. Here is a rough overview:. These acids and the cyclization and oxidation of their backbone form a two-ring chromophore. The two-ring chromophore of GFP absorbs and emits light, e. The chromophore, actually a two-ring chromophore, of GFP lies in the center of a beta-barrel structure. The two-ring chromophore is formed by oxidation and cyclization of the backbone of 3 amino acids: Threonine 65, Tyrosine 66, and Glycine Read on to learn more about GFP, how scientists have evolved this versatile protein to suit their experimental needs, and some of the common applications in the lab.
It has a fluorescent emission wavelength in the green portion of the visible spectrum hence the name , which is due to a chromophore formed from a maturation reaction of three specific amino acids at the center of the protein Ser65, Tyr66, and Gly When first discovered, one of the most surprising aspects of GFP was the fact that the chromophore forms spontaneously and without additional co-factors, substrates, or enzymatic activity — it only requires the presence of oxygen during maturation.
This meant that the protein could be taken directly from A. Victoria and expressed in any organism while still maintaining fluorescence. This tightly-packed structure explains the importance of the entire GFP protein, which is almost completely required to maintain fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable. GFP's main advantage over conventional fluorescent dyes of the time was the fact that it was non-toxic and could be expressed in living cells, enabling the study of dynamic, physiological processes.
Almost as soon as its sequence was elucidated, scientists began engineering new versions of GFP through mutagenesis in order to improve its physical and biochemical properties. In , Roger Y. Tsien described an S65T point mutation that increased the fluorescence intensity and photostability of GFP. This also shifted its major excitation peak from nm to nm, effectively ameliorating the deficiencies found in the wildtype protein and facilitating its widespread use in research. Many other mutations have since been introduced to GFP and new iterations of fluorophores are constantly being engineered.
Table 1 below lists a few common fluorescent proteins and their mutations relative to wildtype GFP. Other scientists studied different fluorescent proteins FPs.
Roger Tsien, a professor at the University of California San Diego , in San Diego, California, reengineered the gene Gfp to produce the protein in different structures. His team also reengineered other FPs. Due to Tsien's and other bioengineers' efforts, GFP could not only exhibit brighter fluorescence, but also respond to a wider range of wavelengths, as well as emit almost all colors, except for red.
Tsien's findings enabled scientists to tag multiple colored GFPs to different proteins, cells, or organelles of interest, and scientists could study the interaction of those particles.
Other laboratories developed fluorescent sensors for calcium, protease and other biological molecules. Since then, scientists have reported more than distinct GFP-like proteins in many species. As GFP does not interfere with biological processes when used in vivo , biologists use it to study how organisms develop.
For example, after , Chalfie and his colleagues applied GFP in the study of the neuron development of C. In a paper, Chalfie and his colleagues describe how they first labeled a specific gene involved in tactile perception in neuron cells with GFP, and then observed the amount of fluorescence emitted by those cells. Because mutant cells produced less or more GFP than normal cells, the abnormal amount of fluorescence production indicated the abnormal development of mutants.
Since then, this field of research expanded to many other organisms, including fruitflies, mice, and zebra fish. Green Fluorescent Protein Green fluorescent protein GFP is a protein in the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light. Ward, and Douglas C. Chalfie, Martin. Davenport, Demorest and Joseph Nicol.
Harvey, Edmund. Luminescence in the coelenterates. Hastings, John, and James Morin. Matz, Mikhail V.
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