Group 9 Project - Fluorescent Proteins
- 1 Introduction
- 2 The Theory of Fluorescence
- 3 The History of the Green Fluorescent Protein
- 4 Green Fluorescent Protein
- 5 Development of New Fluorescent Proteins
- 6 Current research
- 7 Advantages of Fluorescent Proteins
- 8 Limitations of Fluorescent Proteins
- 9 References
- 10 Links to Current Research
- 11 Glossary
The Green Fluorescent Protein (GFP) was first observed in Aequorea victoria a jellyfish in 1962, since then this protein has become a major tool in biological analysis in viewing the many biological processes that were once invisible. The GFP can be bound on many of the thousands of different proteins in living organisms, in real time in vivo and vitro. Many new colours are now available and may be used to view many processes occurring at once for contrast, as well as the creation of many novel viewing tools with even more analysis of cells with many more been made, all this enabling further research in the most common, most dangerous and most understood diseases.
The Theory of Fluorescence
Fluorescence is the spontaneous emission of radiation (ultraviolet, visible or infrared photons) from an excited atom. The the Latin word luminescenz describes all phenomena in which light is released and where a rise in temperature does not occur, whereas incandescence involves heat. The various types of luminescence are classified according to the mode of excitation, fluorescence occurs via the mode of absorption of light to release another wave of light. A prime example is the reaction of luciferase where the oxidation of a substrate by an enzyme releases light.
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There are three principles of fluorescence
1. Excitation; energy/ photons is absorbed by the atom of an appropriate wavelength by the fluorophore which becomes excited.
2. Excited state; the electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, the atom will emit light as the atom's electron reverts to a lower, more stable, energy level, thus fluorescing.
NB; fluorescence is cyclical therefore a fluorophore usually is excited repeatedly.
The fluorescence microscope allows “excitation light” radiate the specimen and then sort out the much weaker emitted light to make up the image. First, the microscope has a filter that only lets through radiation with the desired wavelength that matches your fluorescing material, enabling it to be viewed on a screen.
The History of the Green Fluorescent Protein
Major Milestone Timeline
1565 N. Monardes observed the emission of light by an infusion of wood lignum Nephriticum (first reported observation of fluorescence).
1640 Licetus studied the Bolognese stone and defined it as a non-thermal light emission.
1842 E. Becquerel made the first statement that the emitted light is of longer wavelength than the incident light.
1853 G. G. Stokes introduces the term fluorescence.
1858 E. Becquerel created the first phosphoroscope.
1944 Lewis and Kasha Triplet state
1955 Green fluorescent substance in jellyfish first described.
1962 GFP identified as protein, extracted from Aequorea victoriajellyfish
1979 Shimomura characterized structure of chromophore.
1985 Prasher clones GFP gene from Aequorea victoria.
1993 Structure of GFP chromophore confirmed, flanking amino acid residues corrected from Shimomura's 1979 structure.
2000 onwards The devlopment of GFP variants, as described below in "The Development of New Fluorescent Proteins"
(timeline referenced from here http://www.conncoll.edu/ccacad/zimmer/GFP-ww/timeline.html and the textbook.
GFP and The Nobel Prize
The 2008 Noble Prize in Chemistry "for the discovery and development of the green fluorescent protein" was awarded jointly to:(REFERENCE)
ADD HIS PICTURE HERE Shimomura- When Shimomura first obtained GFP, he did so by squeezing strips of the jelly fish Aequorea through filtercel and then purified it by ammonium sulfate and column chromatography.
ADD HIS PICTURE HERE Martin Chalfie’s experiment by tagging GFP in six individual cells in the transparent roundworm Caenorhabditis elegans, supported previous ideas that it can be used as a molecular tagger or “light bulb”, he also contributed to the ideas of transfecting cells. With his experiment he was able to revolutionize the biosciences.
ADD HIS PICTURE HERE Roger Y. Tsien contribution was that he was able to mutate GFP and create a variety of new colours now termed the NEW FRUIT FP’s, he was also able to make the DsRed FP compact thus more versatile, and finally he created mutants that start fluorescing faster and brighter.
(Reference- The Nobel Prize website)
The Extraction and Purified of GFP from the Aequorea victoria Jelly fish (reference; Green fluorescent protein purification by organic extraction. Protein Expr Purif. 1998 Dec;14(3):382-6. Yakhnin AV, Vinokurov LM, Surin AK, Alakhov YB.;)
1. Transfection into cells via vector for amplification (See Current Research section for detailed methodology)
2. Cell are lysed by sonication, then centrifuged
3. Products are put in ammonium sulfate, ethanol and chloroform and mixed it, resulting in GFP at upper aqueous phase and others proteins at bottom. Alternatively, the hydrophobic nature of the GFP molecule can be utilised for purfication with a size column
4.To confirm the extarction of GFP, the sample is excitated at 395nm (UV light) and checking if a 510nm wavelength emission results.
Further link to purification methods
Green Fluorescent Protein
Structure and Classification
GFP is compromised of 238 amino acids and weighs 26.9kDa, discovered in 1962 by Osamu Shimomura in the jellyfish Aequorea victoria, it glowed green when under ultraviolet light. Its structure was found in 1972 by Shimomura, it is an 11-stranded beta-barrel threaded by an alpha-helix running up the axis of the cylinder, it is predicated that the beta-can structure protects the chromophore and is presumably responsible for GFP’s stability. This protein is now the most commonly used biological marker tool in molecular biology, medicine, and cell biology.
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The Protein Data Bank currently lists 22 GFP’s, they are many mutants, this is done by changing amino acids in them, they actually all have similar structures to aequorea GFP.
The chromophore is in the centre of the beta can and is a 4-(p-hydroxy-benzylidene) imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring.
Tsien (Tsien RY. 1998. The green fluorescent protein. An Rev Biochem 67:509-544.) has classified GFPs into seven major classes based on their spectral characteristics:
(i) Wild-type GFP. The chromophore is in an equilibrium between the phenol and phenolate form. It has two excitation peaks at 395 and 475 nm.
(ii) Phenolate anion. Ser65 has been substituted with Thr, Ala, or Gly. Does not have the 395 nm excitation peak.
(iii) Neutral phenol. Mutation of Thr203 to Ile results in a mutant that only has the 399 nm excitation.
(iv) Phenolate anion with stacked ð-electron system. Mutation of Thr203 to His, Trp, Phe, or Tyr results in yellow fluorescent proteins.
(v) Indole in chromophore. Cyan fluorescent proteins have properties intermediate to those of BFP and EGFP.
(vi) Imidazole in chromophore and phenyl in chromophore. Blue fluorescent proteins have an excitation peak at 383 nm.
(vii) Phenyl in chromophore. It has the shortest excitation wavelength and no apparent uses.
Advantages of GFP
• Applicable to nearly all organisms and in live tissue
• Can be targeted to specific tissues, cells, organelles, or proteins
• Unlikely to diffuse well enough to blur spatial gradients
• Good optical properties: visible excitation, high photo-stability
• Cheap and relatively easy to replicate and distribute
• It’s chromophore does not require a cofactor
• It is highly stable as GFP is resistant to heat, alkaline pH, detergents, photo-bleaching, organic/ non-organic salts, and many proteases.
• Shimomura showed GFP was a relatively small protein which allows little hindrance to the protein or its function. (REFERENCE)
Disadvantages of GFP
• Gene transfection required
• GFP is most efficient temperatures well below 37 °C, thus searches for ones functional in mammalian cells were made.
• GFP only fluoresces in presence of oxygen as the chromophore requires an autocatalytic reaction, thus GFP only works in aerobic environments.
• Some of GFP’s limitations are the slow post-translational chromophore formation.
• Difficulty in distinguishing GFP from background fluorescence when the GFP is not densely localized or highly expressed. (REFERENCE)
Tagging GFP onto Targets
GFP is so versatile and can be tagged by means of labelling reagents with functional groups on amino groups via covalent binding, Wang and Hazelrigg discovered that GFP can be used as a fluorescent tag for the N- or C-termini of proteins. Since there are so many proteins it can belled on nearly all aspects of the cell.
Development of New Fluorescent Proteins
Since the revolutionary development of GFP, extensive research has been conducted with the aim of producing fluorescent proteins (FPs) which cover a broader colour spectrum. These colour mutant FPs are also being genetically engineered to exhibit faster maturation rates, greater photostability, increased brightness, pH insensitivity and reduced oligomerisation and toxicity (Shaner et al. 2007, Shaner et al. 2005). Due to the sheer number of FPs that have been produced, this section will focus on the most current and valuable colour mutant FPs and touch on FPs with other specialisations such as photoactivation and photoconversion.
New FP Colours
Enhanced BFP (EBFP) was one of the first spectral variants engineered from Aquorea GFP, but due to its low brightness and poor photostability it is now unappealing for most research (Shaner, 2007). New blue Aquorea GFP variants Azurite, SBFP2 and EBFP2 show improved brightness and photostability in comparison to EBFP, offering a promising means of imaging live cells in this region (Ai et al., 2007 & Kremers et al. 2007). EBFP2 is the most photostable and brightest blue FP (Shaner et al., 2007).
The cyan FPs (CFPs) began with the production of enhanced CFP (ECFP) from Aequorea GFP (Cubitt et al. 1995). mCerulean followed in 2004 offering a brighter and better general-purpose CFP (Rizzo et al. 2004). In 2006, a monomeric teal-coloured variant mTFP1 was obtained from a Clavularia soft coral protein (Ai et al, 2006). mTFP1 is brighter, less pH sensitive and more photostable than the traditional CFPs, making it an excellent alternative to its predecessors (Shaner et al. 2007).
Most recently, site-directed mutagenesis of ECFP has produced super CFP (SCFP) which is twice as bright as ECFP when expressed in bacteria. SCFP shows promise for use as a fusion tag or as a biosensor for the detection of calcium ion fluctuations, pH changes, metabolites or enzyme phosphorylation (Shaner et al. 2007). Other proteins which fall into this class include mCFPm (Zacharias et al. 2002) and CyPet (Nguyen & Daugherty 2005).
Following the discovery of the original Aequorea GFP discussed previously, many other proteins which express in the green region of the spectrum have been isolated from other Aequorea species, copepods, amphioxus and reef corals (Shaner 2007).
Most of these novel GFPs exhibit no discernable advantage over EGFP (Shaner 2007) and hence will not be discussed. mEmerald, a derivative of EGFP, is currently the best choice for live-cell imaging due to its more efficient folding than EGFP at 37°C (Tsein 1998, Cubitt 1999, Shaner 2007).
Yellow FPs (YFPs) are among the brightest and most versatile probes developed in any of the spectral classes (Shaner 2007). EYFP was developed in 1999 (Miyawaki et al, 1999) and is still widely used despite its high pKa and sensitivity to halides (Shaner et al 2007). EYFP has proved effective in tracking the distribution patterns of single proteins on the membranes of live cells (Ober et al. 2004). mCitrine, derived from the addition of a single mutation in EYFP, is less halide sensitive and twice as resistant to photobleaching as its predecessor (Griesbeck et al, 2001).
mVenus is a popular YFP mutant with a greatly reduced maturation time, however it has low photostability. The fact that mVenus requires only two minutes in vitro or seven minutes in vivo to produce fluorophores makes it ideal for monitoring cellular processes with fast dynamics such as gene expression (Nagai et al., 2002). Super YFP (SYFP), the product of site-directed mutagenesis of EYFP, may hold similar applications as those discussed previously for SCFP. Yellow fluorescent protein for energy transfer, YPet, is the brightest YFP variant. YPet is also reported to have very good photostability and superior acidic resistance to mVenus and other YFP derivatives (Nguyen and Daugherty, 2005).
Compared to the other areas of the spectrum, few probes have been constructed to emit in the orange and red wavelengths. Probes such as DsRed, TagRFP and tdTomato actually have emission profiles in the orange range of the spectrum and not the red range as suggested by their names (Shaner, 2007). mOrange, a member of the ‘Fruits’ series (see ‘Red’ section for details), once dominated this spectrum in terms of brightness, but has average photostability and is unstable at low pH (Shaner, 2007). In an attempt to overcome these problems mOrange2 was engineered from mOrange and shows significantly improved photostability but is still pH sensitive and shows an almost doubled maturation time.
Kusabira Orange (KO), derived as a tetramer from the mushroom coral Fungia concinna, was later modified to give monomer KO (mKO) (Karasawa et al. 2004). mKO demonstrates extremely good photostability and brightness similar to that of EGFP, making it a good candidate for long-term and wide-fluorescence illumination experiments (Shaner 2007 and Shaner 2004)
In 2007 TagRFP was cloned as a dimer from Entacemaea quadricolor sea anemone and appears to be a useful tool for localisation and FRET studies (Merzlyak et al 2007). Random mutagenesis of TagRFP produced the highly photostable, bright and pH resistant TurboRFP.
Many of the problems associated with Discosoma DsRed FP- including slow maturation, an intermediate green state, and tetrameric character- have been the target of many attempted modifications of this protein through both random and site-directed mutagenesis. The production of the monomeric mRFP1 from DsRed was promising, but reduced emission and quick photobleaching still means that it is less useful than monomeric GFPs and YFPs (Campbell et al. 2002)
tdTomato, another of the 'Fruits' proteins, is the brightest of all available FPs, emits at closer to the true red range and is very photostable (Shaner et al 2004). The major drawback in the use of tdTomato is its comparatively large size which is proposed to interfere with fusion-protein packing (Shaner et al 2007).
FPs that emit in the far red area of the spectrum are desirable due to this wavelength of light being less phototoxic and more able to probe deeper into biological tissues (Shaner 2007). The most promising develops have arisen from the site directed mutagenesis of mRFP1 to give monomeric FPs which emit in the 560-610nm wavelength which are collectively referred to as the ‘Fruit’ proteins (Shaner, 2007). Despite their improved emission colours, many Fruits lack the brightness and photostability needed for most experiments (Shaner 2005)). According to Shaner and team, mStrawberry and mCherry are the best reds, with brightness levels of 75% and 50% of EGFP (Shaner 2007). mCherry is more photostable than mStrawberry (Shaner 2005) and as well as a better alternative to mRFP1 for long-term imaging experiments (Shaner 2007).
Developed in 2004, mPlum is one of the first true far-red probes, emitting at 649nm (Wang et al 2004). mPlum has limited brightness (10% of EGFP) but good stability and is recommended for use in multicolour imaging experiments, the imaging of thicker tissues and as a FRET partner for GFPs and YFPs (Shaner 2004, Shaner 2007). Kutushka, a dimeric protein that emits at 635nm, was developed in 2007 and is commercially available from Evrogen as TurboFP635 (Shcherbo et al 2007). Despite being less bright than EGFP, Kutushka has the highest brightness level of any of the FPs in the 650-800nm wavelength area.
mKate (Evrogen, TagFP635) has similar spectral characteristics to Kutushka, brightness on par with mCherry and is reported to be very photostable, making it a good candidate for localisation experiments in this area of the spectrum (Shcherbo et al. 2007). mKate has been reported to exhibit complex photobleaching behaviour which is yet to be well characterised and it is suggested that mCherry remains a more reliable choice for single-molecule imaging (Shaner et al. 2007).
Other Novel FPs
The development of a protein that emits in the infra-red spectrum is the holy grail of fluorescent protein engineering. Accurate in vivo imaging of animal tissue using FPs requires emission wavelengths of 650 to 900nm to minimise absorbance by water, lipids and haemoglobin and to reduce light scattering. Infra-red fluorescent proteins (IFPs) which can be genetically expressed would be incredibly valuable for whole-body imaging in cancer and gene therapy (Shaner 2007).
A paper published in 2009 (Shu et al. 2009) reports the production of a monomeric IFP from a bacteriophytochrome which incorporates biliverdin (an intermediate of heme catabolism) as the chromophore. The IFP was effectively expressed in both mammalian cells and mice, demonstrated the spontaneous incorporation biliverdin and produced infra-red fluorescence.
These proteins display negligible fluorescence until excited by irradiation at a specific wavelength (Lippincott-Schwartz and Patterson 2003). This allows for the highlighting of molecules within a discrete region of a cell as well as a way to study the lifespan and behaviour of proteins independently of other newly synthesised proteins (Lippincott-Schwartz and Patterson 2003). PA-GFP is one such protein that is activated by light of wavelength 488nm and emits in the green wavelength (Wiedenmann et al. 2009). Other FPs which demonstrate “reversible on/off switching” are Dropna, rsFastLime, rsCherry, rsCherryRev and PAmCherry1 (Wiedenmann et al. 2009).
This class of FPs show “light-driven modulation of fluorescence properties” (Wiedenmann et al, 2009) and demonstrate green-to-red conversion when activated at around 400nm (Wiedenmann et al, 2009). These proteins, such as Kaede, KikGR, EosFP, Dendra2, mKikGR, tdEosFP, mEosFP and mEosFP2, are useful for the tracking of fusion proteins, organelles or the fate of embryonic cells during development (Wiedenmann et al, 2009, Shaner et al 2007).
Destabilised GFP variants
These FPs allow the characterisation of the expression timing or lifetime of a target protein due to their rapid turnover by proteolysis resulting in only younger protein chimeras fluorescing (Li et al 1998).
Fluorescent Timer” Protein
This protein, similarly to the destabilised GFP variants, allows the measurement of protein turnover and expression timing (Lippincott-Schwartz and Patterson 2003). It initially fluoresces in the green area of the spectrum before conversion of the fluorophore after several hours leads to emission in the red (Terskikh et al.2000). Using the ratio of green to red fluorescence allows the age of the tagged protein to be determined.
Future Engineering Endeavours
Criteria for future engineering endeavours as proposed by Shaner et al 2005:
• Improved brightness
• Monomeric structure
• High contrast
• Uncomplicated photoconversion
• Reversible photoactivation
• Red-to-green photoconversion
• Improved expression in the far-red or near-infrared regions
Current research in cell biology with fluorescent proteins is a vast field of study. We shall focus our investigation on a specific technique that has significant clinical consequences. This technique uses spliced fluorescent genes to create transgenic cells capable of producing fluorescent proteins. The principle behind this technique is based on the model for protein synthesis and thus an explanation of the protein synthesis model is an essential requisite.
Protein synthesis occurs in 3 stages:
The first stage: (Transcription)
1) Inside the nucleus of a cell, the double stranded DNA helix unwinds in the area of the gene containing the information for synthesizing a specific protein.
2)The enzyme RNA polymerase moves along the unwound DNA strand at the location of the gene using it as a template to create a complementary strand known as a messenger RNA strand (mRNA). This complementary strand is made by linking RNA nucleotides that are complementary to the gene nucleotides on the DNA strand. Note: In any RNA strand, the base thymine is replaced with uracil and the start codon (AUG) and a stop codon (UAA or UAG or UGA) control the length of the strand.
3)The mRNA strand then undergoes modifications in the nucleus to contain only the base sequence that will code for the protein. Most genes contain non-coding regions known as introns and coding regions called exons. Thus its complementary mRNA strand will also contain coding and non-coding regions. The non-coding regions are excised by splicing introns from the mRNA and joining the exons together.
The second stage: (Amino acid activation) This stage occurs in the cell cytoplasm where the aminoacyl-tRNA synthetise enzyme attaches amino acids to transfer RNA molecules (tRNA). Each type of amino acid is attached to its specific tRNA.
The final stage: (Translation)
1)The mRNA strand binds onto a ribosome in the cytoplasm at the end with the start codon (AUG).
2)A tRNA molecule binds to the start codon on the mRNA within the ribosome.
3)A second tRNA binds to the next codon of the mRNA within the ribosome.
4)The first tRNA is released from the ribosome and the ribosome moves along the mRNA strand one codon at a time. Two tRNAs are temporarily bound within a ribosome at any one time and their amino acids linked together forming a polypeptide chain which after further modifications becomes the final protein.
A transgenic organism is one whose genome contains genes from another species. The aim of creating a transgenic organism is to obtain a favorable characteristic in the organism’s phenotype. This desired characteristic of phenotype is obtained by altering an organism’s normal genotype to include the gene from another species coding for the desired characteristic. Once a desired gene is incorporated into an animal’s genotype, the process of protein synthesis allows the transgenic organism to express this new characteristic in its phenotype.
In the case of fluorescent research, the gene responsible for fluorescence phenotypic characteristics in one organism can be spliced and inserted into the genotype of another organism for experimentation. The recipient organism is then able to produce the fluorescent proteins (coded for by the inserted gene) in its phenotype which makes it an efficient tool for targeting specific proteins, tissues and organs of interest.
The production of a fluorescent transgenic organism involves several steps summarized below:
1)Organisms containing fluorescent proteins are identified. The chromosome and the gene coding for the fluorescence are identified in the organism.
2)A cell sample is taken from the organism and the gene of interest is isolated from its DNA strand. This involves “cutting” it out of its DNA strand using enzymes called restriction endonucleases. The restriction endonucleases cut DNA at specific site so the desired gene can be removed from the DNA strand. The cut ends are known as “sticky ends”.
3)The gene is then incorporated into a recipient organism’s DNA. To do this, the recipient organism’s cell DNA must first be cut with the same restriction endonucleases at a desired location (usually just before the stop codon at the end of a gene coding for a particular protein). The foreign gene is then inserted with a promoter sequence by microinjection or vector transfer into the fertilized egg cell or a somatic cell of a recipient.
4)The inserted foreign gene will be attracted to and connect at the recipient’s cut DNA in a process called “annealing”. The DNA is now known as recombinant DNA as it contains a combination of the recipient’s normal genes as well as foreign genes.
5)DNA ligases are added to the annealed DNA fragments to help strengthen the bonds of the new recombinant DNA. DNA ligases are sealing enzymes found in all living organisms that help make and repair DNA.
6)As the cell develops and its genetic information read, the recombinant DNA will not only code for the production of the normal protein, but will also code for the production of the fluorescent protein that will be attached to the normal protein. This will be true of the recipient cell and all its descendants.
The creation of transgenic organisms with the capability to produce fluorescent proteins has significant clinical consequences particularly in the understanding of inexorable diseases such as cancer, malaria and prion infections.
Cancer is the leading cause of death worldwide claiming approximately 8 million people in 2009. The WHO estimates this figure to rise to 12 million by 2030. In Australia, cancer claims approximately 42,000 lives every year and an estimated 114,000 new cases are expected in 2010. A cancer or malignant neoplasm is a class of disease in which cells display uncontrolled mitotic divisions, invasion of surrounding tissues and metastasis (spread via the circulatory or lymphatic system). Cancer cells are unspecialized and have no other function other than reproduction. These cells invade tissues and organs diminishing their functioning gradually until failure.
Fluorescent proteins provide a means by which cancer can be studied in vivo. The implications of this are significant as it allows researchers to study the characteristics of this disease as it develops in the body over time. A histologically intact primary tumour from a cancer patient is obtained by surgical means and transfected a specific fluorescent protein. The transfected tumour cells are then transplanted into immunodeficient transgenic mammals (usually mice) which have been genetically modified to display a different fluorescent protein. This creates a contrast between the healthy cells of the recipient organism and those of the tumour cells as both display different coloured fluorescence. The fluorescent proteins fluoresce for the whole life of a cancer cell and after cell division, fluoresce it both the parent and the daughter cells. Using this contrast, researchers are able to visualize, in real time, important aspects of cancer in living animals, including tumour cell mobility, invasion, metastasis, angiogenesis and to investigate the efficacy of anti-cancer therapeutics.
This technique of studying cancer has so far yielded some interesting results. GFP expressing tumour cells revealed that angiogenesis occurred early in tumour formation when as little as 60–80 tumour cells were present inducing vasodialation and changes to blood vessel morphology (12,13). This could potentially unearth critical information on the ability of tumour cells to recruit blood vessels that vital for their growth and spread. Research by Yamauchi and Yamamoto even explored the mechanism by which tumour cells migrate down through narrow capillaries. By expressing red fluorescent proteins in the tumour cell cytoplasm and green fluorescent proteins in the cell nucleus they showed that tumour cells are highly motile and capable of changing their morphology to great extents in order to squeeze down narrow vessels. This is critical information on the metastatic ability of cancer cells and explains to some degree the mechanism by which cancer cells are able to spread to body extremities.
Malaria is the world’s most serious infectious disease and is responsible for one million deaths annually. There are currently between 250 – 500 million people affected by malaria most of them in developing countries. In Africa, malaria is an especially serious problem where a child dies from it every 30 seconds. In Australia, malaria was declared eradicated in 1981 but approximately 700 - 800 cases occur each year from travellers infected elsewhere with northern Australia the receptive zone for malaria transmission. Malaria is a parasitic infection caused by four species of the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. The infection begins when Plasmodium sporozoites enter human blood through the bite of an infected female Anopheles mosquito. The Plasmodium first enters the liver cells, where it multiplies, and then red blood cells, where it continues to proliferate. When each infected red blood cell bursts, multiple new parasites are released which are capable of infecting new red blood cells repeating the process. Growing resistance to anti-malarial medicines such as chloroquine and mosquito pesticides has spread very rapidly undermining malaria control efforts across the globe.
Fluorescent proteins provide a unique method of imaging the entire malaria parasite lifecycle from development to transmigration inside both insect and vertebrate hosts. This is achieved by transfecting the malarial parasites with genes encoding for green-fluorescent protein (GFP) or red-fluorescent protein (RedStar) creating a transgenic organism (19). Fluorescent microscopy can then be used to visualize the parasites behaviour inside the entire mosquito vector and cells of the vertebrate host. Using this technique, Rogeiro et. al found that Sporozoites are formed inside oocysts at the mosquito midgut wall. They are released into the hemolymph and eventually bind to the salivary glands, which they then invade. Then when the mosquito bites its host, the sporozoites are released into the host’s circulatory system (34).
Another research focusing on control of malaria found that transgenic mosquitoes were less susceptible to plasmodium infection and had fewer sporozoites in their salivary glands than control mosquitoes. They also found transgenic mosquito parasite transmission was reduced by more than twofold. The team found this to be a result of the expression of the SM1 peptide in the mosquito midgut that severely reduced vector competence by inhibiting Plasmodium development. This has paved the way for further experiments where multiple gene modifications in mosquitoes are used as a malaria control measure. Other research has focused on breeding experiments where fluorescent proteins are used to differentiate male and female mosquitoes. Since female mosquitoes only mate once in their lifecycle and are the transmitters of the malaria parasite, sterile males (marked by fluorescent gonads) are sorted and released into the environment where they mate with the infected females. Since the female will be unable to reproduce, the malaria parasite life cycle is ended.
A prion is an infectious protein that has been altered from its normal shape to an abnormal shape. Stanley Prusiner discovered prions as a new class of infective agents in the 1970’s. Motivated by the death of a patient from Creutzfeldt Jacob disease (CJD), he experimented with these infective agents and found that radiation tests designed to cause mutations in nucleic acids had no effect on prions and thus he concluded that prions did not contain nucleic acids (DNA or RNA). This conclusion made prions even more mysterious as nucleic acids are the molecules replicated during cell division and reproduction, thus how prions reproduce is an area of great interest in cell biology.
A prion has the capability to convert other similar but normal proteins into an abnormal prion shape. Whenever a prion comes in contact with a normal prion protein, it causes the normal protein to 'flip' into an abnormal shape, thereby becoming a prion. Any other normal prion protein that a prion touches will also be converted, creating a domino effect. Prions can be transmitted from one organism to another causing disease in healthy organism. In the 1990s “mad cow disease” (spongiform encephalopathy) became epidemic in cattle in the United Kingdom killing 200,000 cattle and also 150 people who consumed infected cattle products. The people who consumed the infected cattle products also developed spongiform encephalopathy. Some other diseases caused by prions include scarpie in sheep, fatal familial insomnia, Gerstmann-Straussler-Scheinker Syndrome and chronic wasting disease.
The study of prion diseases provides an excellent opportunity for fluorescent proteins to help understand the expression of prion proteins, misfolding of prion proteins and cell death from the action of prion proteins. The method involves fusing fluorescent proteins to prions or to normal proteins that will be subjected to prions. The characteristics and behaviour of prions can then be observed under fluorescent microscopy. In one study by Barmada and Harris, scarpie prions were inoculated in mice and tagged with enhanced green fluorescent protein (EGFP). As the disease progressed in the mice, the researchers noted that these fluorescent tagged prions accumulated in the neuropil, axons and Golgi apparatus of neurons allowing them to hypothesize the mechanisms by which prions work. Another study by Alfred Goldberg showed that neurons with prions in their cytosol, were less efficient in breaking down fluorescently marked proteins when compared to uninfected neurons. This sheds some light on the inhibitive capability of prions on neurons and possibly how they cause central nervous system diseases.
Advantages of Fluorescent Proteins
Some advantages in using fluorescent proteins include:
• The imaging of structures, cells, tissues, organs and even living organisms in vivo.
• There is no known adverse disruption to the normal functioning of the cell biochemistry.
• Fluorescent proteins are easily obtained from fluorescent organisms mainly corals, jellyfish and marine invertibrates.
• The isolation and transfection of fluorescent proteins is a higly successful process that produces minimal hazardous wastes unlike other fluorescent techniques like radioactive dyes and fluorescent nano dots.
• FP's are available in a vast array of colours covering different emission spectrum for imaging different tissues.
Limitations of Fluorescent Proteins
Problems associated with FPs- from Wiedenmann et al. 2009 and Shaner et al 2005:
• Cytotoxicity due to FP
• Environmental sensitivity
• Light-induced cytotoxicity
• Potential biological activity exerted by FPs
• No strict criteria under which the FPs are tested- results are often biased and therefore not trustworthy for researchers
• Commercial availability/price
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Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein Ste´phanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo. VOLUME 23 NUMBER 1 JANUARY 2005 NATURE BIOTECHNOLOGY.
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References for The Development of New FPs
Ai, H, Henderson, JN, Remington, SJ & Campbell , RE. (2006). Directed evolution of a monomeric, bright and photostable version of clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochemistry Journal, 400(3), Retrieved from http://www.ncbi.nlm.nih.gov/sites/entrez doi: PMCID: PMC1698604
Ai, H, Shaner, NC, Cheng, Z, Tsien, RY & Campbell, RE. (2007). Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry, 46, 5904-5910.
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877-7882.
Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A. and Tsien, R. Y. (1995). Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448-455.
Cubitt, A. B., Woolenweber, L. A. and Heim, R. (1999). Understanding structure function relationships in the Aequorea victoria green fluorescent protein. Methods Cell Biol. 58, 19-30.
Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. and Tsien, R. Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein: Mechanism and applications. J. Biol. Chem. 276, 29188-29194.
Karasawa, S., Araki, T., Nagi, T., Mizuno, H. and Miyawaki, A. (2004). Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381, 307-312.
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Merzlyak, E. M., Goedhart, J., Shcherbo, D., Bulina, M. E., Shcheglov, A. S., Fradkov, A. F., Gaintzeva, A., Lukyanov, K. A., Lukyanov, S., Gadella, T. W. J. et al. (2007). Bright monomeric red fluorescent protein with an extended fluorescent lifetime. Nat. Methods 4, 555-557.
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Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. and Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell biological applications. Nat. Biotechnol. 20, 87-90.
Ober RJ, Ram S and Ward ES, 2004. Localization accuracy in single-molecule microscopy. Biophys J vol 86 pp 1185-1200. Nguyen, A. W. and Daugherty, P. S. (2005). Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355-360.
rizzo et al 2004
Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E. and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572.
Shaner, N. C., Steinbach, P. A. and Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nat. Methods 2, 905-909. Shaner NC, Patterson GH and Davidson MW, 2007. Advances in fluorescent protein technology Journal of Cell Science 120, 4247-4260.
Shcherbo, D., Merzlyak, E. M., Chepurnykh, T. V., Fradkov, A. F., Ermakova, G. V., Solovieva, E. A., Lukyanov, K. A., Bogdanova, E. A., Zaraisky, A. G., Lukyanov, S. et al. (2007). Bright far-red fluorescent protein for whole-body imaging, Nature Methods vol. 4, pp. 741-746.
Shu X, Royant A, Lin MZ, Aguilera TA, Lev-Ram V, Steinbach PA & Tsien RY, 2009. Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome, Science, vol. 324, pp. 804-807.
Terskikh A, Fradkov A, Ermakova G , Zaraisky A, Tan P , Kajava AV, Zhao X , Lukyanov S, Matz M, Kim S, Weissman I & Siebert P,‘" Fluorescent timer": protein that changes color with time’, Science, vol:290 iss:5496 pp. 1585-1588 Tsien, R.Y. The green fluorescent protein. Annual Review Biochemistry, vol. 67, 509–544 (1998).
Wang, L., Jackson, W. C., Steinbach, P. A. and Tsien, R. Y. (2004). Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 16745-16749.
Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).
Links to Current Research
Chromophores; Molecules which are able to absorb light
fluorochromes or fluorophores; Molecules which are able to absorb and emit light Filtercel;
FRET; Fluorescence Resonance Energy Transfer
Photobleaching; the destruction of fluorophores by light
Random mutagenesis; random incorporation of mutations in DNA
Site-directed mutagenesis; targeted insertion of a mutation in a DNA sequence, usually by use of of a plasmid vector
Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer