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WO2007019382A1 - Proteines de type gfp fluorescentes/chromoproteines photoactivables et applications d'imagerie - Google Patents

Proteines de type gfp fluorescentes/chromoproteines photoactivables et applications d'imagerie Download PDF

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WO2007019382A1
WO2007019382A1 PCT/US2006/030592 US2006030592W WO2007019382A1 WO 2007019382 A1 WO2007019382 A1 WO 2007019382A1 US 2006030592 W US2006030592 W US 2006030592W WO 2007019382 A1 WO2007019382 A1 WO 2007019382A1
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proteins
protein
green
red
fluorescent
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Anya Salih
Mikhail Vladimirovitch Matz
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University of Sydney
University of Florida
University of Florida Research Foundation Inc
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University of Sydney
University of Florida
University of Florida Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to fluorescent and colored proteins and their use. These materials and methods are particularly advantageous for labeling and detection technology. Specifically exemplified are colored and/or fluorescent proteins isolated from marine organisms that have photoactivatable properties. These proteins offer a wider array of colors and photoinducible properties compared to existing wild-type green fluorescent protein (GFP), its modified variants or anthozoan GFP-type proteins utilized in current labeling and detection technology.
  • GFP green fluorescent protein
  • the most commonly used optical biosensing method is to monitor intracellular species with fluorescent labels.
  • the dominant technology for such in vivo labels, cellular assaying, imaging, detection and advanced drug, gene and protein screening is currently based on the organic fluorescent molecules and the green fluorescent protein (GFP) and the super-family of related GFP-type proteins are the most widely used fluorescent labels.
  • GFP green fluorescent protein
  • isolated from the Pacific Northwest jellyfish (Aequorea victoria) has become an important reporter marker for non-destructive analysis of gene expression. It is unique as its fluorescence is encoded by a single gene, whose expression in any organism or cell produces fluorescence.
  • GFP revolutionized the study of cellular processes in vivo, allowing direct genetic encoding of fluorescence and has become the most powerful microscopic, imaging and molecular tool in cell biology, medicine and biotechnology.
  • GFPs aid the study of study of cells - what is in them, how they use proteins to communicate and perform various functions and how these processes are affected by diseases.
  • GFP can be attached to a protein of interest and visualized to study the manipulation of targets, genes and proteins.
  • the impact of GFPs and related proteins on science, medicine and biotechnology is due to their unique genetic encoding that enables fluorescence expression and consequent real-time and non-invasive reporting in living cells.
  • the protein fluorescent characteristics and no other co-factors are required, a case which is relatively unique in nature because other pigment proteins (e.g. rhodopsins, flavins, phycobilins, etc) require many enzymes and co-factors for the colour characteristics to be cellularly expressed.
  • GFPs are also highly stable in cells, are very bright and are non toxic.
  • GFP genetic sequence provides a basis for cell-based monitoring of GFP- linked targets upon administration of external drugs.
  • GFP has also acted as a marker of protein dynamics and behavior in cell biology.
  • GFP can not only be used in early stage target characterization but also in retrieving non-invasive 'whole organism' data in studies utilizing, for example, live mice with transfected GFP-tagged tumors, in drug activity trials. These applications have been translated to drug discovery where, GFP, and its color variants have been utilized in fluorescence and confocal imaging, drug screening assays and in in vivo diagnostics. Accordingly, GFP has become a versatile fluorescent marker for monitoring a variety of physiological processes, visualizing protein localization and detecting the expression of transferred genes in various living systems, including bacteria, fungi, and mammalian tissues.
  • a range of in vivo fluorescence labeling and detection technologies were originally based on a single (wild-type) GFP. Since its discovery, numerous modifications have been made to alter its spectral properties, to provide for significant enhancement in fluorescence intensity and enabling simultaneous multi-colour imaging of several proteins, genes or cells. These additions to the portfolio include blue-fluorescent, cyan-fluorescent, and yellow-fluorescent proteins.
  • wild-type GFP wt-GFP
  • the chromophore is formed in an autocatalytic, post-translational cyclization and oxidation of the tripeptide unit.
  • GFP has an 11 -stranded beta-can with a central alpha helix in which the chromophore, 4-( ⁇ -hydroxybenzylidene)-5- imidazolinone is encased.
  • the chromophore forms autocatalytically from the tripeptide Ser-Tyr-Gly in an oxidation reaction.
  • the wt-GFP absorbs blue light at 395 nm and emits green light at 508 nm.
  • New fluorescent protein-based assays have the potential to improve drug discovery by elucidating signaling pathways, protein translocation, and other vital processes.
  • fluorescence-based cellular assays such as fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), and fluorescence correlative spectroscopy (FCS).
  • FRET fluorescence resonance energy transfer
  • FLIM fluorescence lifetime imaging
  • FCS fluorescence correlative spectroscopy
  • Such assays will potentially provide a variety of supplementary data and validated cell lines expressing fluorescent proteins will increase the versatility of fluorescent-protein based cellular assays by providing more targets for measuring drug
  • Spectral deconvolution has been used recently to image and separate the signals from several different fluorescent proteins.
  • the limitations of spectral discrimination can place an upper limit on the number of resolvable probes that can be developed by rational or random mutagenesis techniques.
  • a range of coral and other marine invertebrate fluorescent proteins, with a range of colors covering the visible spectrum and possessing a suit of unique optical properties, have provided the opportunity for expanding the life-sciences and medical fluorescence technologies.
  • Red fluorescent proteins are especially in high demand because they are low energy and are, therefore, less phototoxic. Additionally, background fluorescence from cellular tissue, cell components and light scattering are markedly reduced in the red spectral region.
  • fluorescent proteins FPs
  • GFP-like proteins existed only in bioluminescent organisms, it was relatively recently found that the coloration of the non-bioluminescent animals, such as corals, is due to a diverse family of GFP-type proteins (Matz et al. 1999, 2002; Lukyanov et al. 2000; Gurskaya et al. 2001; Dove et al. 2001; Labas et al 2002). GFPs from these animals offer an exciting potential to expand the use of the available GFP-type proteins in optical applications, such as multi-label imaging, single and multi-photon microscopies and in the development of labels for in vivo tracking of cellular dynamics and fluorescent molecular sensors.
  • GFPs the anthozoan RFPs - the fluorescent protein drFP583, commercially named DsRed
  • DsRed See, for example, Matz et al. 1999 Nature Biotechnology; Labas, Y. A., N. G. Gurskaya, Y. G. Yanushevich, A. F. Fradkov, K. A. Lukyanov, S. A. Lukyanov and M. V. Matz. 2002, Diversity and evolution of the green fluorescent protein family, Proc Natl Acad Sci USA 99:4256- 4261).
  • DsRed-type RFPs have been described, with a broad emission spectrum at 570-610 nm but there is a still a strong demand for novel RFPs with superior optical properties.
  • DsRed like most other coral proteins, is tetrameric, this property can cause protein aggregation and cell toxicity.
  • Optimization of protein molecular properties has led to the development of monomelic proteins, and there is a great demand for spectrally and optically diverse GFP monomers.
  • Other improvements over GFP have included increased brightness (quantum yield), photostability, temperature and pH stability, suppression of photodynamics (flickering) and an even further extension of the range of emission wavelengths.
  • Photoactivation is a phenomenon of light-induced optical change in a protein. Photoactivation behaviour was first reported in a wild-type jelly-fish-derived GFP, which upon intense illumination with UVA light increased in fluorescence three-fold upon excitation at 488 nm. The fluorescence is a result of photoconversion of the fluorophore population via a shift from neutral phenols to anionic phenolates (see, Heim, R., et al. Proc. Nat.
  • UV light causes rapid and intense enhancement of fluorescence upon irradiation.
  • One variation is genetically modified to be highly suitable for analyzing protein kinetics within cells (Patterson, G. H. and Lippincott-Schwartz, J. (2002) A Photoactivatable
  • Photoactivatable fluorescent proteins represent a novel effective tool to selectively mark various proteins, organelles, and cells in vivo and to monitor their movement in real time. Another such photoactivatory or phototransforming process, the "greening" of
  • GFP-like proteins leads to a red-to-green color shift following irradiation and was first described for wild-type GFP and subsequently for DsRed.
  • DsRed from a reef anthzoan has emission maxima at green 509 nm (weak) and red 583 nm (strong). Its synthesis includes one more stage to that of green FPs, involving the extension of the conjugated 7T-system of chromophore - dehydrogenation of the bond between the alpha-carbon and amino nitrogen of the first chromophore-forming residue (Gross L A, Baird G S, Hoffman R C, Baldridge K K 5 Tsien R Y. Proc Natl Acad Sci USA.
  • DsRed greening is caused by the selective bleaching of the red species of the tetramere by light, usually multi-photon wavelength, causing the fluorescence of the green species, which is normally quenched by Forster resonance energy transfer (FRET), to be enhanced.
  • FRET Forster resonance energy transfer
  • the "greening" property of DsRed is used as a powerful technique for regional optical marking of cells and proteins.
  • DsRed like most other coral proteins, is tetrameric, this property can cause protein aggregation and cell toxicity. Optimization of protein molecular properties has led to the development of monomeric proteins, and there is a great demand for spectrally and optically diverse GFP monomers.
  • the covalent structure of the chromophore of the green-to-red converters is different to wt-GFP and all contain a chromophoric unit that derives from the tripeptide His-Tyr-Gly (Mizuno et al. 2003 MoL Cell 12, 1051-1058).
  • Kaede and Eos have been utilized in optical marking as they undergo a stable green to red conversion on irradiation by UV or violet light, visualized by imaging at excitation by green light.
  • Such light inducible proteins can be used as reporters for molecular dynamics where a small cell area is color-changed by irradiation by UVA light.
  • chromoproteins which possess a unique type of photoconversion: initially nonfluorescent, they are purple-blue in color (causing purple coloration of anemone tentacle tips) and convert to red fluorescence (i.e. kindle) after irradiation by intense green light (Lukyanov et al 2000 Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275, 25879-25882; Gurskaya, N.G. et al. (2001) GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507, 16-20).
  • the first such protein was isolated from Anemonia sulcata, named asulCP, and possessed excitation-emission maxima are at 575 and 595 nm, respectively (Lukyanov et al. 2000; Gurskaya et al. 2001; Chudakov, D. M., Belousov, V. V., Zaraisky, G., Novoselov, V. V., Staroverov, D. B., Zorov, D. B., Lukyanov, S. & Lukyanov, K. A. (2003) Kindling fluorescent proteins for precise in vivo photolabeling. Nature Biotech. 21, 452-452; United States Patent Application No. 20030092884, Lukyanov, Sergey A.
  • chromoproteins In these chromoproteins, cyclization leads to formation of a six-member rather than five-member ring, creating the extended conjugated vr-system by breakage of the polypeptide chain immediately before the chromophore (Martynov V I, Savitsky A P, Martynova N Y, Savitsky P A, Lukyanov K A, Lukyanov S A. J Biol Chem. 2001, 276:21012-21016).
  • photoactivatable fluorescent proteins are especially useful for direct labeling and tracking of intracellular and intercellular protein movement, important in many applications, including scientific research, drug development and diagnostics.
  • the conventional imaging technique to track intracellular protein movement is photobleaching. During fluorescence recovery after photobleaching (FRAP), a selected area of interest (AOI) in a cell or organelle, containing a fluorescent marker
  • fluorescently tagged protein/s e.g., fluorescently tagged protein/s
  • bleached i.e., fluorescently quenched
  • the movement of the fluorescently labeled protein can be analyzed by following the recovery of fluorescence in the bleached AOI.
  • fluorescence loss in photobleaching (FLIM) protein movement can be analyzed by monitoring a loss of fluorescence in an unbleached cell part, as fluorescently tagged proteins leave a bleached region and thereby reduce fluorescence in an unbleached region.
  • FLAP fluorescent localization after photobleaching
  • photobleaching by intense light can be very damaging to cells and alter their physiology, structures, 8 UOS-IOOXCl protein kinetics and making interpretation of photobleaching experimental results prone to artifact. Since photobleaching is an indirect method of monitoring protein kinetics, an improved method that would allow direct means of monitoring protein movement is required. Photoactivatable fluorescent proteins enable direct protein monitoring and present a vastly improved method to that of FRAP and FLIM.
  • the labeling strategies provided by these proteins represent "second-generation" tools for fluorescence tracking applications, in the sense that they can be used to directly track objects of interest after photolabeling, rather than indirectly monitor movement via fluorescence recovery into a photobleached region, as with traditional fluorescence recovery after photobleaching-based approaches.
  • Photolabeling techniques using photoactivatable proteins are superior to FRAP and FLIM techniques and include the use of photoactivatable PA-GFP, of the greening properties of DsRed by photobleaching, of the green-to-red photoconverting properties of the coral protein Kaede and similar proteins, and of the kindling of non- fluorescent CPs to a red fluorescent state. Since many of these proteins exhibit certain short comings such as extremely low quantum yield, low photostability, aggregation, etc., the search for GFP-like proteins with novel photoproperties is a focus of great scientific interest.
  • Light-induced activation can also take the form of photochromism (photoswitching) in which the protein switches between dim and bright states by irradiation with light of appropriate wavelengths.
  • photochromism photoswitching
  • the process is also linked to the fluorescence behavior of wild-type GFP with its two absorption maxima (395 nm and
  • FPs and CPs display complex photophysical properties, revealed by single-molecule techniques, exhibiting intense emission intensity variations (on-off blinking behavior). They can often be 9 UOS-IOOXCl photoreversibly switched between light and dark states at time scales ranging from microseconds to hours (Pierce et al. Nature 1997; Dickson et al Nature 1997, Haupts et al PNAS 1998; Schwille et al. PNAS 1999, Lounis et al 2001; Heikal et al. Chem Phys 2001' Nifosi et al 2003).
  • Protein optical devices based on bacteriorhodopsin (BR) are known, but do not allow for fluorescence detection.
  • the photochromic materials can be used in single- molecule optical storage (See, W. E. Moerner, Science 265:46 (1994)).
  • Optical memory storage devices based on photochromism of wt GFP fluorescent proteins have been suggested (United States Patent 6,046,925, Tsien , et al. 2000).
  • the present invention provides coral fluorescent protein (FP) or chromophoric proteins (CPs), their optical properties and methods for the utilization of these properties, especially proteins from Acropora millepora, including, for example, 2 cyans (amilFP489 and amilFP496), 2 greens (amilFP502 & amilFP512), red (amilFP593) and purple-blue chromoprotein (amilCP588) and their applications.
  • FP coral fluorescent protein
  • CPs chromophoric proteins
  • the subject invention describes photoactive properties for various proteins, red-to-green conversion and kindling, as well as their photochromicity - reversible photoswitching properties, hi one embodiment it has been found that the quantum yield of kindled protein (amilCP588) is high, photoswitching is efficient, and there is also involvement of greening in the same protein.
  • the subject invention provides new and useful applications of the green converter and kindling proteins such as their uses as ROS sensors.
  • FIG. 1 is a graph showing the fluorescence excitation & emission spectra of cloned proteins from Acropora millepora (amilFP489, amilFP502, amilFP512, amilFP593 and amilCP624).
  • UOS-IOOXCl is a graph showing the fluorescence excitation & emission spectra of cloned proteins from Acropora millepora (amilFP489, amilFP502, amilFP512, amilFP593 and amilCP624).
  • FIG. 2 Table of spectral properties (excitation max., emission max., molar extinction coefficients, quantum yield) of proteins in live tissues and of cloned proteins from A. millepora.
  • FIG. 3 shows phototransformation of red FP (582 nm) in live cells of Goniopora tenuidens on blue light irradiation.
  • FIG 4 Cyan amilFP489 and green cloned and puried proteins from A. millepora undergoing rapid intensification of fluorescence upon irradiation by UVA- violet light; red protein amilFP593 shows no change.
  • FIG 5 Shows reversible on-off switching by UVA (activation) and blue light (deactivation) of photoactive green amilFP502 immobilized in polyacrilamide gel.
  • FIG. 6 shows the photoinduced greening of red amilFP593.
  • FIG 7 Shows rapid photoinduced greening of red amilFP593 by 514 nm laser line scanning to produce bright and stable yellow-green color.
  • FIG 8 shows reversible on-off switching by UVA (deactivation) and green light (activation) of photoactive red amilFP593 air-dried as thin film on glass.
  • FIG 9 Yellow-to-Green (Y-to-G) converter from Euphyllia ancora.
  • FIG. 10 shows kindling of amilCP588 by green light.
  • FIG. 11 shows excitation and emission spectra of kindled protein.
  • FIG. 12 kindling and red to green conversion of amiCP588. Greening is by reversible photo-bleaching.
  • FIG. 13 shows multi-colour emissions of amilCP588 after exposure to various excitations, including UV, blue, and red light.
  • FIG. 14 shows kindling by 2-photon irradiation at 800 nm.
  • FIG. 15 shows lifetime components of kindled and photoconverted amilCP588.
  • FIG. 16 shows amilCP588 used as a ROS-cellular sensor illustrating spectra of the protein without exposure to H 2 O 2 (noH 2 O 2 ), after exposure to 100 nM OfH 2 O 2 , and after exposure to 10 ⁇ M OfH 2 O 2 in an in vitro preparation.
  • the present invention provides coral fluorescent protein (FP) or chromophoric proteins (CPs), their optical properties and methods for the utilization of these properties, especially proteins from Acropora millepora, including, for example, 2 cyans (amilFP489 and amilFP496), 2 greens (amilFP502 and amilFP512), red (amilFP593) and purple-blue chromoprotein (amilCP588) and their applications.
  • FP coral fluorescent protein
  • CPs chromophoric proteins
  • Biochemical optical alterations involve the interaction of two or more residues of the protein and external agents such as molecular oxygen give rise to the colored and/or fluorescent feature of the proteins.
  • Light-induced alterations involve modification of their optical properties via biophysical, biochemical or molecular means to produce photoactivation, photoswitching or photoconversion.
  • the use of these proteins facilitate real-time detection in vivo, a substrate is not required, and the relatively small size make the proteins very advantageous.
  • Their reversible photoactivatory/photoswitching/photoconversion behavior can dramatically improve analyses with no loss of fluorescence during repeated imaging and allow selective labeling and tracking, providing a much greater detailing of spatio-temporal events.
  • FIG.l and 2 show excitation and emission spectra, molar extinction coefficients and quantum yields of proteins from A. millepora. These proteins exhibit light-inducible properties such as photoactivation, photoswitching, green-to-red conversion, red-to-green conversion and kindling that form the basis of the present invention (the polynucleotide sequences that encode these proteins are disclosed in U.S. Patent Publication No. 2005-0048609, and patent application Serial No.
  • Photoactivatable proteins are proteins which, upon light irradiation, instead of bleaching, alter their optical properties - increase their fluorescence absorption, emissions and excitation and/or alter their color.
  • the proteins of the subject invention can be used, for example, in molecular fluorescent tagging whereby a protein of interest is fused with a fluorescent protein of the subject invention.
  • the product of such a gene shows the functional characteristics of the protein of interest, but bears the fluorescent label allowing tracing its movements. See, for example, Eichinger, L., S. S. Lee and M.
  • the colored and fluorescent proteins of the present invention can be detected using standard long-wave UV light sources or optical designs appropriate for detecting agents with the excitation/emission characteristics of the proteins exemplified herein. They can be photoactivated by using the standard instruments and methods, including natural sunlight, epifluorescent widefield, confocal, multiphoton, TIRF and various types of spectroscopic irradiation. These proteins are referred to herein as “detectable proteins”, “marker proteins”, “photoactivatable”,
  • the proteins of the subject invention can used in labeling approaches in which they are irradiated by specific wavelengths, photoactivated and thereby allow direct induction of the fluorescent signal of different intensity or color similar to such proteins as PA-GFP (Patterson G.H. and Lippincott-Schwartz, J. A photoactivatable GFP for selected photolabeling of proteins and cells. Science 297, 1873-1877, 2002) or KFP and asCP - kindling proteins (Chudakov, D. M., Belousov, V. V., Zaraisky, G., Novoselov, V. V., Staroverov, D. B., Zorov, D. B., Lukyanov, S. & Lukyanov, K. A.
  • One aspect of the invention pertains to desirable optical behavior of Acropora millepora, and including proteins of A. nobilis, A. hyacinthus, Acropora aculeus, Goniopora djiboutiensis, Montipora efflorescencs, Fungia danai, Stylocoeniella armata, Pontes pontes, Echinophyllia echinata, Mycedium elephantotus (the polynucleotide sequences that encode these proteins are disclosed in U.S. Patent Publication No. 2005-0048609 and patent application Serial No. 11/058,952; filed
  • the proteins of the subject invention can be used.
  • the proteins can be used to identify cells.
  • the proteins can be used to express fluorescence in a cell.
  • One use for this method is in pre-labeling isolated cells or a population of similar cells prior to exposing the cells to an environment in which different cell types are present. Detection of fluorescence in only the original cells allows the location of such cells to be determined and compared with the total population.
  • a second group of methods concerns the identification of cells that have been transformed with exogenous DNA of interest. Identifying cells transformed with exogenous DNA is required in many in vitro procedures as well as in in vivo applications such as gene therapy.
  • the reversible photoactivatory/photoswitching/photoconversion property of disclosed proteins can be employed for repeated imaging and selective labeling and tracking of organelles, cells, proteins and molecules, providing a spatio-temporal 15 UOS-IOOXCl capability. Repeated imaging becomes possible since these proteins, instead of irreversibly bleaching upon repeated irradiation often necessary during imaging, show enhancement of fluorescence, in some cases by over 300-fold.
  • FRET-Fluorescence Resonant Energy Transfer This technique allows observation and quantification of molecular interactions. It commonly requires two fluorescent donor and acceptor proteins. Currently the most widely used pair is CFP and YFP (mutated variants of GFP); the photoactive proteins of the subject invention may be substituted for either or both of them.
  • Photoactive proteins described herein can be used as components of the photochromic FRET (pcFRET) system to form a reversible light-driven switch.
  • pcFRET photochromic FRET
  • Photoactivating the photoswitching protein alters its absorption spectrum. For example, green light at 561 run leads to switching on of amilCP588 altering absorption max. to ⁇ 600nm, while UVA light at 405 run switches off the kindled protein and alters absorption max. to 560 nm.
  • a donor spectrally selected to overlap the switched off spectrum of photoactive protein will have its own fluorescence quenched in the switched off state of that protein.
  • a donor fluorophore can be selected to match the absorption spectrum of the switched on state of the photoswitchable protein (in the case of amilCP588 abs. Max. -600 nm).
  • FRET can be switched on and off by photoactivation of the photoswitching protein.
  • spectrally separable proteins include epifluorescence, confocal microscopy, multi-photon microscopy, fluorescence lifetime imaging microscopy (FLIM), total internal reflection fluorescence microscopy (TIRFM), near-field scanning optical microscopy, flow cytometry, and fluorescence activated cell sorting (FACS) using modular flow, dual excitation techniques as well as a wide range of spectroscopic techniques, including various types of ensemble and single molecule and kinetic and stead state spectroscopies.
  • FLIM fluorescence lifetime imaging microscopy
  • TRFM total internal reflection fluorescence microscopy
  • FACS fluorescence activated cell sorting
  • TIRFM Total internal reflection fluorescence microscopy
  • the proteins of the subject invention can be used in FRET applications, when fluorescence intensity or lifetimes (FLIM microscopic imaging) or TIRF imaging are employed.
  • photoactivable GFPs in which light exposure causes fluorescence enhancement. These include the wild-type wtGFP in which irradiation by UV (at about 365nm) causes local fluorescence enhancement and photoactivatable PA-GFP with 504 nm excitation and 517 nm emission, obtained by mutagenesis of wtGFP. PA-GFP photoactivation is achieved by light in the range of 350-450 nm.
  • GFP see Patterson, G. H. & Lippincott-Schwartz, J., A photoactivatable GFP for selective photolabeling of proteins and cells., Science 297: 1873-1877, 2002
  • KFPs Choudakov et al. 2002, 2003
  • PA-CFP Choudakov, D.M. et al. 2004.
  • UVA-induced intensification of fluorescence generally occurred subsequent to prior rapid (lms to lsec pulse or lmin to several minutes) irradiation by blue light (460-490 nm epifluorescent or 488 nm confocal laser scanning), followed by UVA or violet irradiation (e.g., 405 laser) (see FIG. 5).
  • Protein fluorescence emission intensity increased dramatically after even only one second of UVA irradiation. This was frequently followed by a rapid further increase for about the first 5 minutes of irradiation, a more gradual increase for about 30 to about 60 minutes by about 30% to about and over 100% when subsequently viewed by epifluorescence microscopy or imaged by single- or multi-photon excitation.
  • GFP-like proteins such as PA-GFP (Patterson & Lippincott-Schwartz, 2002) or PS-CFP (Chudakov et al. 2004) and KFP (Chudakov et al. 2003) the proteins are stable, form bright states, do not require very intense irradiation that can be toxic to cells, and show rapid photoactivation, achievable in ms to sec to min.
  • millepora proteins exhibit reversible photoswitchable behaviour - irradiation at one wavelength (e.g. blue 488 nm laser line) rapidly quenches fluorescence, while irradiation with another wavelength (e.g., epifluorescent UVA at 340 to 390, or violet 395 to 460 nm; or 405 nm laser line) increases fluorescence by 100-300% or more.
  • Imaging of fluorescence intensity can be conducted by a 'reading' or 'neutral' wavelength (for example, but not limited to, at 458, 476 or 514 nm laser lines).
  • millepora cyan amilFP489, excitation ⁇ 441nm / emission 489 nm; cyan amilFP496 excitation 477 / emission 496 nm; green amilFP502, excitation -448 nm/ emission
  • the proteins retain photoswitchable characteristics in dried protein form, immobilized in films such as PVA film and in gels (for example, agar gel), in solution (for example, phosphate buffer at pH 7.2) and frozen state. These proteins retained their switching property after numerous switching cycles at room temperature or at 46 0 C.
  • the response time of switching of proteins varied in the millisecond to second to minutes range and with a repeatability of 10 to >100 times, exhibiting bright and stable fluorescence in switched on form. Fluorescence intensity in switched off state was at 80% and down to 5% of the original fluorescence intensity at ensemble protein level. Photoswitching can also be done at single molecule level by immobilizing the protein in thin films or gels.
  • Photoswitching is not limited to the above switching on or switching off wavelengths and can include all visible or multi-photon/infra-red irradiation.
  • photoactivated (kindled) fluorescent form of amilCP588 showed photoswitching to a dim state by UVA irradiation (or 405 nm laser line) as well as by blue light, and converted to a bright form at green light irradiation.
  • Photoswitching of the "greened" form of amilFP594 occurred by UVA-violet off-switching and green light on- switching (Fig. 6-8 greening and photoswitching) similar to amilCP588. This process is reverse to that occurring in cyan and green proteins.
  • Imaging without causing photoactivation can be done by 458 nm laser line (i.e., imaging or reading wavelength).
  • the photoswitching mechanism of UVA activation and blue light deactivation is unknown but is likely to be related to the formation of the switched protonated form converting to a bright deprotonated form of the chromophore as in wt-GFP and PA-GFP.
  • the kinetics of photobleaching and photoreactivation are similar in both amilFP593 and amilCP88 and are fitted by a two-exponential function, indicating a similarity in the mechanisms and the presence of multiple components in both.
  • Exemplary embodiments of the present invention include several proteins that can be activated by light from about 350 nm to about 450 nm and also by blue light over about 450 nm.
  • UOS-IOOXCl The ability to be activated by blue light over about 450 nm 20 UOS-IOOXCl enables photoactivation by laser lines at about 458 nm and about 488 nm, which are common to confocal microscopes.
  • increases of fluorescence emission intensity of photoactivated proteins range from about 2-5 fold up to about 100-200 fold.
  • Some embodiments of the proteins include green FPs such as PA-GFP (emitting at about 512-517 nm) and also shorter greens
  • the photoactivatory, photochromic/photoswitching properties at the ensemble level in solution, gels or thin films or at single-molecule level can be implemented in the field of biological imaging.
  • the photochromic properties of the proteins can be used for direct optical labeling to follow diffusion or transport of proteins, particles, signaling molecules in live cells.
  • Cellular dynamics of molecules of interest can be analyzed by activating the fluorescent proteins to their fluorescent state.
  • photoswitching fluorescent proteins to proteins of interest the dynamics can be studied in living cells.
  • molecular-scale bionsensors can be formulated.
  • the photoinduced switching property of fluorescent proteins is usable in the formulation of nondestructive read-out systems.
  • the photoswitching property at the ensemble level in solution, gels or thin films or at single-molecule level can be implemented in the field of opto- electronics where it can provide molecular-scale devices.
  • Imaging with 'writing' wavelength can be done, using a pre-determined writing wavelength.
  • Photoinduced effects can be captured confocally and analyzed as spectral, intensity or lifetime changes as described.
  • Miniature laser sources and detectors that have recently become commercially available can be used to photoactivate various proteins in multilayer assemblies in gels or films to produce the required on/off and reading optical signals.
  • Red-to-green photoconvertible proteins are photoconvering proteins from scleractinian corals that possess two or more stable optical states, that following the irradiation with light of appropriate wavelengths, altered their absorption, excitation or emission spectra i.e., photoconverted from one wavelength to another wavelength. Formation of a red emitting stage from the green via a biochemical / molecular reaction. Red-to-green change is caused by intense photo-bleaching of red emitting stages and increased fluorescence from green stages.
  • One method of the invention involves rapid light-inducible "greening" of red coral proteins from several coral species, including A. millepora's amilFP593 (Fig 6- 8 show greening of amiIFP593) avoiding prolonged or very intense irradiation, including tissue proteins of Acropora hyacinthus, A. nobilis, Pontes porites, Porites cylindrica, Goniopora tenuidens, Lobophyllia hemprichii, L. pachysepta, Favia pallida, yellow-orange protein from Euphyllia ancora, and red coral proteins disclosed in U.S. Patent Publication No.
  • Three-photon excitation selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction of fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the "greening" effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 hours without affecting cell viability.
  • This technique allows individual cells, organelles, and fusion proteins to be optically 22 UOS-IOOXCl marked and has utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population.” (Merchant et al. 2001).
  • red-to-green converting proteins are described, including amilFP593 and amilCP588 (Fig. 12).
  • the amilFP593 acquires a shoulder at 532 nm during greening which at maximal greening rates (achieved by 561 nm pulse or several seconds of 514 nm irradiation) increases in brightness while the main red (593nm) peak disappears.
  • These proteins are photoconverted not only by multi-photon by also by visible light (blue, green and red) and by UV, greatly expanding the technological applications of green-to-red converters.
  • the parameters that induce the color change of these proteins in methods of the present invention are greatly simplified over those of DsRed and demonstrate applications that show the utility of this optical highlighter.
  • the resultant converted green FP has high quantum yield and is stable.
  • the mechanism of colour conversion appears to be similar to DsRed red-to-green colour change and may relate the selective bleaching of red acceptor versus green donor chromophores and cessation of the protein's intramolecular FRET.
  • FIG. 10 shows kindling and FIG. 11 excitation and emission spectra of kindled protein and FIG. 12 shows red to green conversion of amiCP588.
  • Greening can be achieved by reversible photoconversion when low irradiation intensities of 405 nm or 488 nm laser line are used.
  • the mechanism of greening is unknown but it can be hypothesized that the greening mechanism is also similar to DsRed-type selective bleaching.
  • Another method of the present invention results in "reddening" of proteins from several coral species, including Favia pallida, Favites sp, Trachyphyllia sp,
  • A. hyacinthus protein and green A. millepora protein amilFP512 The mechanism appears to be distinct to that of Kaede-type conversion (see Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., and Miyawaki, A., An optical marker based on the UV-induced green-to-red-photoconversion of a fluorescent protein., PNAS 99: 12651- 12656, 2002) since amilFP512 lacks the chromophoric unit that derives from the tripeptide His-Tyr-Gly (Mizuno et al. 2003) known to be necessary for such color change.
  • the resultant dim red color following irradiation of amilFP512 may involve selective bleaching of green fluorophore, leaving the red fluorophore with reduced fluorescent quantum yield.
  • Another method of the present invention involves activating non-fluorescent chromophoric proteins by various light wavelengths to convert them from a non fluorescent to a highly red fluorescent state.
  • the kindled proteins emit at about 600 nm to about 650 run and are maximally excitable by green or orange light at about
  • Exemplary proteins include those from A. hyacinthus and A. m ⁇ llepora, specifically amilCP588 (which has absorption at about 588 mm in its non- fluorescent state and emission maximu at 624 nm in kindled state) (FIG. 10-11).
  • the described proteins particularly amilCP592 and other A. millepora blue, green and red FPs, have the property of changing their lifetimes following photoactivation, kindling, photoconversion and photobleaching by various light wavelengths from UV to red and infrared (multi-photon). The lifetimes become significantly longer indicating that intramolecular FRET (energy transfer) is inhibited possibly upon bleaching of acceptor fluorophores as in DsRed.
  • FRET energy transfer
  • FIG. 10, 11 shows kindling of amilCP588.
  • the protein amilCP588 is highly stable when kindled, can be stored at room temperature or frozen or dried in the dark for prolonged periods, emitting in red wavelengths at about 624 nm of high quantum yield at green light excitation.
  • the protein amilCP588 therefore does not require very strong irradiation to achieve kindling, unlike asFP595 (Chudakov et al. 2002, 2003) that needs very intense irradiation, which can be deleteriously to living cells. It can be kindled rapidly at intensities of ⁇ 1000 micro Enstein per meter square per second (i.e. ⁇ 300 W/m 2 ). 25 UOS-IOOXCl
  • AmilCP588 is very bright when kindled, with quantum yield (Fig. 2) significantly higher than that of other known kindling chromophoric proteins or of red far fluorescent proteins (for example, KFP quantum yield is only 0.04 and extinction coefficient 80,000 M-I cm-1) (Chudakov et al. 2003).
  • amilCP588 has a relatively high absorbance (molecular absorption coefficient), thus having highly efficient light energy conversion and can be used as an acceptor within the donor-acceptor pairs for FRET applications.
  • the chromoprotein amilCP588's polynucleotide sequence, as well as chromoprotein of A. hyacinthus, is the subject of U.S. Patent Publication No.
  • green light is the maximal kindling wavelength, including epifluorescent as well as laser lines at about 514 rim, about 543 nm, and about 561 nm.
  • the chromoproteins are also kindled by weak UV, blue and red light, unlike the other known kindled protein asFP595 from Ammonia sulcata (also known as asCP) which is kindled by intense green light and not by other wavelengths (see U.S. Patent Publication Nos. 2002-
  • the amilCP588 is unique also because UVA or blue light irradiation causes colour conversion similar to DsRed-type red to green colour change which results due to selective quenching of red Fluorescence (FIG. 12). Following stabilization of maximal red 624 nm emissions (due to kindling by green light), further irradiation causes a decrease of the 624 nm peak and a progressive increase of the green emissions imaged (at about 498 nm to about 534 nm) at UVA or blue excitation. The peaks vary depending on excitation and bleaching wavelengths. The resultant green fluorescence is stable and bright (see FIG. 4). AmilCP588 therefore, shows significantly different quenching characteristics to that shown by asCP (asFP595).
  • amilCP588 can be bleached or quenched by intense or prolonged irradiation by especially UV (e.g. 405 laser line) or by other wavelengths.
  • UV e.g. 405 laser line
  • FIG. 13 shows multi-colour emissions of amilCP588 after exposure to various excitations, including UV, blue, and red light.
  • short exposure to UVA irradiation of kindled protein results in red color with maximum 26 UOS-IOOXCl emission at about 601 nm at blue excitation.
  • More intense UVA irradiation produces orange emissions at about 580 nm to about 585 run at blue excitation, which are also produced by intense irradiation at longer duration by other wavelengths.
  • Combined irradiation by intense UVA and other selected wavelengths e.g.
  • amilCP588 is readily kindled by 2-photon irradiation (760-900 nm) which makes it very suitable for whole-body imaging. For example, using deep penetrating infrared wavelengths to kindle labeled components in deep tissue.
  • Fig. 14 shows kindling by 2-photon irradiation at 800 nm. 2-photon irradiation causes rapid kindling suitable for whole body imaging.
  • Another characteristic of amilCP588 is its photoresponsive lifetime kinetics.
  • amilCP588 protein shows a multi-component lifetime decay, with ns and ps components. Their proportions change significantly in response to the irradiating wavelengths (FIG. 15).
  • amilCP588 protein has the advantage of multi- wavelength kindling, unlike asCP and KFP, which are limited to green light kindling. While amilCP588 is most rapidly kindled by green wavelengths using epiftuorescent excitation or laser lines (e.g. at about 514 nm, about 543 nm or about 561 nm), amilCP588 can also be kindled by UV to red wavelengths. UV or blue light irradiation causes colour conversion similar to DsRed-type red to green color change.
  • amilCP588 can also be converted to yellow, orange or green forms depending on the level of irradiation enabling multi-colour imaging and levelling by using a single protein. Morevoer, amilCP588 has 2 emission peaks at UV to blue light excitation: weaker green and stronger red kindled emissions. This also enables 27 UOS-IOOXCl imaging in dual channels rather than one for KFP as well as ratiometric imaging.
  • amilCP588 exhibits low kindling laser intensities. In contrast, asCP is kindled by high intensity green laser line; this is often detrimental to live cells, causing bleaching of fluorophores or cell damage. amilCP588 can be kindled at low laser power, thus enabling imaging at more appropriate physiological conditions.
  • amilCP588 At 50% laser power excitation of Leica confocal microscope, kindling occurs within seconds but requires longer irradiation to reach maximal intensity at 90-120 seconds duration.
  • amilCP588 also exhibits a desirable lack of blue-light quenching. In contrast, asCP is rapidly 'switched off or quenched by irradiation by blue light. Lack of such blue-light quenching in amilCP588 enables imaging at blue light wavelengths, commonly used in GFP-type cell imaging, without switching off protein fluorescence. Blue light, however, does cause a decrease of red kindled emissions with appearance of green fluorescence.
  • the amilCP588 can be "bleached" or "switched off (see FIG. 3) by any high intensity irradiation.
  • amilCP588 shows profound fluorescence lifetime differences following different levels of protein photoactivation, kindling, and levels of bleaching by different light wavelengths.
  • the lifetimes of kindled amilCP588 are some of the fastest of GFP-type proteins, down to picoseconds compared to the usual nanosecond rates.
  • FIG. 15 shows lifetime component differences of kindled amilCP588.
  • amilCP588 an excellent ROS sensor.
  • One embodiment of the present invention is a membrane-specific molecular label.
  • amilCP588 is often localized within membranes and neurites in anthozoans. It is therefore likely that it may, via its oligomerisation tendency or due to specific groups attached to the protein that provide it with membrane binding capacity, be developed as a membrane or nerve-specific live cell fluorophore.
  • photoconvertible GFPs of the present invention including amilFP593 and amilCP588, show several spectral responses upon exposure to different conditions, elements and compounds (e.g. changes in pH and levels of CO2) and can be used as cellular environmental sensors.
  • the photoconvertible GFP-like proteins of the present invention show spectral changes after photoactivation upon exposure to reactive oxygen species (ROS), hydrogen peroxide and other oxidants, and reactive nitrogen species (RNS) present at cellular concentrations. These can therefore be used as redox or reactive oxygen species (ROS) cellular sensors.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • Selection of amino acid substitutions to reduce tetramerization can be done by site-directed mutagenesis using standard methods well-known to one of ordinary skill in the art can be used to disrupt the two tetramerization interfaces of amilFP593, amilCP588 and FPs and CPs described herein. Various substitutions at each position can be made, and fluorescence evaluated as described in known literature. Origomerization of variants can be determined using standard techniques, e.g., analytical ultracentrifugation (Laue and Stafford, 1999; Baird et al. 2000) and velocity sedimentation can be used to ascertain which forms are present (monomers, dimers and/or tetramers), and tequilibrium sedimentation will be used to measure the oligomer association constants.
  • analytical ultracentrifugation Laue and Stafford, 1999; Baird et al. 2000
  • velocity sedimentation can be used to ascertain which forms are present (monomers, dimers and/or tetra
  • Polynucleotides cDNA sequences encoding the proteins of the present invention are provided in the Matz et al patents documents ,composed of either RNA or DNA, and preferably, the polynucleotides are composed of DNA. Specifically exemplified are DNA sequences that encode for Acropora millepora FPs and CPs as well as other species listed. Sequences of the subject invention may utilize codons preferred for expression by the selected host strains. These sequences may also have sites for cleavage by restriction enzymes, and/or initial, terminal, or intermediate DNA sequences which facilitate construction of readily expressed vectors. The subject invention also concerns variants of the polynucleotides that encode detectable proteins. Variant sequences include those sequences wherein one or more nucleotides of the sequence have been substituted, deleted, and/or inserted. 30 UOS-IOOXCl
  • Polynucleotide molecules containing DNA sequences encoding the photoactive CPs or FPs of the present invention can be introduced into a variety of host cells including bacterial cells, yeast cells, fungal cells, plant cells and animal cells. Methods by which the exogenous genetic material can be introduced into such host cells are well known in the art and are as described in the Matz et al. patent documents.
  • a polynucleotide sequence encoding a marker protein of the subject invention is fused to a DNA sequence encoding a selected protein in order to directly label the encoded protein.
  • Expressing such a fluorescent and/or colored protein in a cell results in the production of labeled proteins that can be readily detected. This is useful in confirming that a protein is being produced by a chosen host cell. It also allows the location of the selected protein to be determined. Cells that have been transformed with exogenous DNA can also be identified without creating a fusion protein.
  • the method relies on the identification of cells that have received a plasmid or vector that comprises at least two transcriptional or translational units.
  • a first unit encodes and directs expression of the desired protein, while the second unit encodes and directs expression of the detectable protein.
  • Co-expression of the detectable protein from the second transcriptional or translational unit ensures that cells containing the vector are detected and differentiated from cells that do not contain the vector.
  • a gene sequence is generally fused to one or more DNA sequences that encode proteins having defined amino acid sequences and the fusion proteins are expressed from an expression vector. Expression results in the production of fluorescent proteins of defined molecular weight or weights that may be used as markers (following calculation of the size of the complete amino acid sequence).
  • Amino acid replacements that produce different color forms permit simultaneous use of multiple reporter genes.
  • Different colored proteins can be used to identify multiple cell populations in a mixed cell culture or to track multiple cell types, enabling differences in cell movement or migration to be visualized in real time 31 UOS-IOOXCl without the need to add additional agents or fix or kill the cells.
  • Photoactive, photoconverting and photoswitching proteins increase the color palette available as for example, is seen for different color emissions achieved as a result of reversible and partial bleaching of kindled amilCP588 or of red-to-green color conversion by amilFP593.
  • Photoactive proteins described herein enable improved ability to analyze dynamics of tagged proteins, organelles, molecular signals, or other substances and their subcellular localization in a spatially and temporally defined manner by photoinduced switching to fluorescent state or by photoinduced color conversion.
  • the subject invention concerns polynucleotides comprising an in-frame fusion of nucleotide sequences encoding multiple genetic markers.
  • a polynucleotide of the invention may comprise a first nucleotide sequence that is operably linked in-frame to a second nucleotide sequence.
  • the polynucleotide encodes the amino acid sequences of the detectable protein and another genetic marker such that the genetic markers are in direct contact with one another, i.e., where the last amino acid of the fluorescent genetic marker is immediately contiguous with the first amino acid of the other genetic marker, or they can be separated by a peptide linker sequence, for example, as described in U.S. Pat. Nos.
  • the pigmented tissues were dissected and immobilized on slides under glass coverslips or were frozen at -2O 0 C or -75 0 C. Irradiation was conducted using standard techniques of epifluorescence and confocal microscopies, using epifluorescent or laser line irradiation at different wavelengths of UVA and visible spectrum (see FIG. 3 phototransformation of red FP (582 mm) in live cells of Gonipora tenuidens on blue light irradiation).
  • Photoactivation of cellular and of cloned FPs/Cps were performed as follows: pigmented ectodermal tissues were dissected from live samples collected from the reef or aquarium-kept corals, or from chemically fixed or air-dried samples, mounted on a coverslip. Purified proteins were dissolved in phosphate buffer and their photoactivatory properties were analyzed in solution, or dried onto glass coverslips, or immobilized on coverslips that have been coated with polyethylene glycol (PEG) polymer chains or immobilized in a gel matrix (e.g. agarose gels) or other immobilizing medium such as polymers (e.g., poly vinyl alcohol, poly electrolytes, viscous liquids or studied in cell cytoplasm.
  • PEG polyethylene glycol
  • Fluorescence intensity or spectra were imaged and analyzed using fluorescence, confocal (405, 458, 476, 488, 514, 543, 561, 633 nm laser lines) and multi-photon excitation (760 to 950 nm) and spectroscopic instruments, following irradiation at these various wavelengths. Irradiation was tested at different time intervals, from lmillisec to 1 s to 1-3 hours to 24 hours. Imaging was done using conventional imaging techniques (for example, Leica TCS SP2 confocal microscope with spectroscopic capability).
  • Photoactivation of cloned proteins was done on metal-affinity purified proteins from bacteria, dissolved in IxPBS-Na (0.15M NaCl, 0.05 M Na2HPO4, pH 7.4) buffer and mounted in agar, gels or air-dried as thin films on glass. Irradiation was at UVA epifluorescent light, or confocal laser lines of 458, 476, 514, 488, 543 and 561 nm as well as 2-photon wavelengths as described above (objective xlO or x20).
  • Additional useful applications of the technology described herein include, but are not limited to, photochromic fluorescent proteins and optical memory storage devices. Methods which are well known to those skilled in the art can be used to utilize photoswitchable proteins as potential recording media for optical storage of information. As described in US patent 6,046,925 photochromic fluorescent or 34 UOS-IOOXCl chromophoric protein can be used in single-molecule optical storage formulations or for multi-molecule optical storage. Information can be stored by switching between states of the photochromic fluorescent protein moiety by irradiation.
  • Reading can be achieved, for example, by irradiating with low intensities or by irradiationg with wavelength that do not elicit a photoswitching response, example - 458 nm wavelength can be used as reading wavelength with photoswitchable amilFP593, together with 561 nm as writing, and 405 nm as erasing wavelength.
  • writing and erasing can be achieved, for example, by irradiating with high intensities.
  • it can be necessary to refresh a state by reading the state of the photochromic fluorescent protein moiety and rewriting with high intensity irradiation.
  • Other approaches of utilizing coral photoactive proteins are as described in US patent 6,046,925, for example in near-field optical storage or in holographic optical storage.
  • the novelty here is the fastly improved brightness of coral photoactive proteins, specifically of amilCP88 and amilFP593; their speed of photoswitching, repeatability of photoswitching events and stability, compared to jelly-fish derived protein US patent 6,046,925.
  • ROS reactive oxygen species
  • ROS include hydrogen peroxide (H 2 O 2 ), superoxdie radical and other oxidants. Also relevant are the reactive nitrogen species (RNS).
  • RNS reactive nitrogen species
  • HyPer H 2 O 2 sensitive green fluorescent protein probe
  • the described proteins particularly amilCP592 and other A. millepora blue, green and red FPs, have the property of changing their lifetimes following photoactivation, kindling, photoconversion and photobleaching by various light wavelengths from UV to red and infrared (multi-photon). The lifetimes become significantly longer indicating that intramolecular FRET (energy transfer) is inhibited possibly upon bleaching of acceptor fluorophores as in DsRed.
  • FRET energy transfer
  • the photoswitching and photoconvertible GFP-like proteins of the present invention including amilFP489, amilFP502, amilFP497 5 amilFP512, amilFP593 and amilCP588, show ROS dose-dependent alteration of their optical properties following light activation on exposure to ROS.
  • Tested ROS included H 2 O 2 , rose bengal diacetate which is generator of singlet oxygen, as well as nitric oxide.
  • the changes include partial or total quenching of fluorescence of photoactive proteins when irradiated by light in the presence of cellularly relevant ROS concentrations.
  • Photoactive proteins show ratiometric alteration of their green, yellow and red emissions during light induced photoconversion.
  • photoconverting wavelengths e.g., 405, 488, 514, 543 nm laser lines, or multi-photon irradiation, etc
  • the green-yellow emissions increase while red components decrease in proportion to H2O2 and nitric oxide concentrations, with sensitivity at micromolar to nanomolar levels (Fig. 16).
  • the relative absorption and excitation spectra show ROS-dose specific alteration.
  • the speed of certain types of photoconversion such as the red-to-green conversion of photoactive proteins is accelerated in proportion to H 2 O 2 concentration.
  • light induced rates of red-to-green conversion of amilFP593 and of the kindled amilCP588 are accelerated by x3-7 orders of magnitude compared to control preparations without H 2 ⁇ 2 or with antioxidants added (e.g. horseradish peroxidase).
  • ROS/NOS reactive oxygen species
  • these probes provide the capability to monitore production of specific types of ROS.
  • a powerful multifunctional ROS sensor is created.
  • ROS sensitive fluorescent probes provide more sensitive measurement of singlet oxygen, the ability to take measurements in live cells over real time and the ability to combine the fluorogenic probes with other fluorogenic or fluorescent probes, in order to simultaneously investigate multiple parameters of live cellular function.
  • photoactive proteins are as markers or labels or indicators for oxidative damage due to storage of stored foods.
  • the subject photoactive proteins may be incorporated into a variety of different compositions of matter, where representative compositions of matter include: food compositions, synthetic compositions, pharmaceuticals and cosmetics to monitor oxidation levels.
  • ROS and oxidation levels of biological fluid samples may also be monitored using individual or sets of photoactive/photoswitching proteins.
  • Biological samples include, e.g., blood, plasma, serum, cerebral spinal fluid, urine, amniotic fluid, interstital fluid, and synovial fluid.
  • Solid biological samples include, e.g., a tissue, cells, tissue culture, fixed cells, cell supernatants, or even portions (or extracts) of tissue or cell matter.
  • Biological samples may also be a urine, saliva, tears, mucus secretions, sweat, blood (or blood products), tissue, feces or other biological samples suspected of having oxygen radicals.
  • any suitable method of detection is useful in detecting fluorescent or colored probes of this invention. Because ROS, NOS, pH, CO2 alter fluorescence characteristics, the quantitative detection of intracellular concentration in intact living cells or tissues or whole organisms (e.g., mice) can be performed by fluorescent intensity measurements, using fluorescence microscopy, confocal or multi-photon microscopy, FLIM, TIRF, flow cytometry, polarization microscopy, frequency domain fluorescent tomography and other imaging technologies.
  • the photoactive chromoproteins and fluorescent proteins of the subject invention can be used in sunscreens, as selective filters, etc., in a manner similar to the uses of the proteins described in WO 00/46233. Distinct from it, the present invetion involves the use photoactive proteins as photodissipating
  • FRET arrays FRET arrays.
  • Our research has shown that FPs and CPs in corals are tuned for efficient energy transport (Salih et al. Nature 2000; Salih et al. SPIE 2004, 2006) via 38 UOS-IOOXCl radiative transfer and FRET (Gilmore et al. 2003 Photochem Photobiol; Cox & Salih, 2006).
  • Corals have adapted to use GFP-based FRET arrays to effectively dissipate excessive solar energy. Photoactivation, photoconversion, photoswitching and kindling processes enhance the sunscreening properties of FPs/CPs. By mimicking organization of coral proteins in vivo to create solar energy dissipating formulations, heterogeneous mixtures can form effective sunscreens.
  • kits comprising in one or more containers and a polynucleotide and/or protein of the present invention.

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Abstract

La présente invention concerne une protéine fluorescente (FP) de corail ou des protéines chromophores (CP), leurs propriétés optiques et des procédés d'utilisation de ces propriétés, en particulier des protéines d'Acropora Millepora, comprenant par exemple deux cyanes (amilFP489 et amilFP496), deux vertes (amilFP502 et amilFP512), une chromoprotéine rouge (amilFP593) et bleu-pourpre (amilCP588) et leurs applications.
PCT/US2006/030592 2005-08-03 2006-08-03 Proteines de type gfp fluorescentes/chromoproteines photoactivables et applications d'imagerie Ceased WO2007019382A1 (fr)

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US20140288270A1 (en) * 2013-03-22 2014-09-25 National Taiwan University Isolated chromoprotein of stichodactyla haddoni
CN106404211A (zh) * 2016-11-09 2017-02-15 哈尔滨工程大学 一种不依赖激发光强的上转换荧光强度比测温方法
CN106500864A (zh) * 2016-11-09 2017-03-15 哈尔滨工程大学 一种使用高功率激光泵浦的上转换荧光强度比测温方法

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8647887B2 (en) 2009-01-29 2014-02-11 Commonwealth Scientific And Industrial Research Organisation Measuring G protein coupled receptor activation
WO2013163681A1 (fr) * 2012-05-01 2013-11-07 University Of Western Sydney Protéines fluorescentes et leurs utilisations
US20140288270A1 (en) * 2013-03-22 2014-09-25 National Taiwan University Isolated chromoprotein of stichodactyla haddoni
CN106404211A (zh) * 2016-11-09 2017-02-15 哈尔滨工程大学 一种不依赖激发光强的上转换荧光强度比测温方法
CN106500864A (zh) * 2016-11-09 2017-03-15 哈尔滨工程大学 一种使用高功率激光泵浦的上转换荧光强度比测温方法
CN106404211B (zh) * 2016-11-09 2018-12-25 哈尔滨工程大学 一种不依赖激发光强的上转换荧光强度比测温方法
CN106500864B (zh) * 2016-11-09 2019-01-29 哈尔滨工程大学 一种使用高功率激光泵浦的上转换荧光强度比测温方法

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