Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items

Abstract

Isolated and purified nucleic acids encoding green fluorescent proteins from Renilla reniformis and the green fluorescent protein encoded thereby are also provided. Mutants of the nucleic acid molecules and the modified encoded proteins are also provided. Compositions and combinations comprising the green fluorescent proteins and/or the luciferase are further provided.

1 . An isolated substantially purified  Renilla reniformis  green fluorescent protein (GFP) encoded by an isolated nucleic acid molecule encoding a  Renilla reniformis  green fluorescent protein, comprising a nucleic acid sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto.  
     
     
         2 . A mutein of the GFP of  claim 1  that exhibits altered spectral properties.  
     
     
         3 . A mutein of the GFP of  claim 1  that exhibits a reduced tendency to form multimers.  
     
     
         4 . A composition, comprising the green fluorescent protein of  claim 1  and at least one component of a bioluminescence generating system.  
     
     
         5 . The composition of  claim 4 , wherein the bioluminescence generating system is selected from those isolated from: an insect system, a  coelenterate  system, a  ctenophore  system, a bacterial system, a  mollusk  system, a  crustacean  system, a fish system, an  annelid  system, and an earthworm system.  
     
     
         6 . The composition of  claim 4 , wherein the bioluminescence generating system is selected from those isolated from: fireflies,  Mnemiopsis, Beroe ovata, Aequorea, Obelia, Vargula, Pelagia, Renilla, Pholas Aristostomias, Pachystomias, Poricthys, Cypridina, Aristostomias, Pachystomias, Malacosteus, Gonadostomias, Gaussia, Watensia, Halisturia,  Vampire squid,  Glyphus, Mycotophids, Vinciguerria, Howella, Florenciella, Chaudiodus, Melanocostus,  Sea Pens,  Chiroteuthis, Eucleoteuthis, Onychoteuth is, Watasenia, cuttlefish, Sepiolina, Oplophorus, Acanthophyra, Sergestes, Gnathophausia, Argyropelecus, Yarella, Diaphus, Gonadostomias  and  Neoscopelus.    
     
     
         7 . A mutein of  claim 2 , comprising substitution in amino acids at amino acids 56-75 of SEQ ID NO. 27, whereby the spectral properties are altered.  
     
     
         8 . The composition of  claim 7 , wherein the bioluminescence generating system is selected from those isolated from  Aequorea, Obelia, Vargula  and  Renilla.    
     
     
         9 . A combination, comprising: 
 an article of manufacture; and    a  Renilla reniformis  green fluorescent protein (GFP) encoded by a nucleic acid molecule encoding a  Renilla reniformis  green fluorescent protein, comprising a nucleic acid sequence that encodes the protein of SEQ ED NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto.    
     
     
         10 . The combination of  claim 9 , further comprising: 
 at least one component of a bioluminescence generating system, whereby the combination is a novelty item.    
     
     
         11 . The combination of  claim 10 , wherein the component of the bioluminescence generating system comprises a  luciferase.    
     
     
         12 . The combination of  claim 10 , wherein the component of the bioluminescence generating system comprises a  luciferin.    
     
     
         13 . The combination of  claim 9 , wherein the article of manufacture is selected from among toys, fountains, personal care items, fairy dust, foods, textile and paper products.  
     
     
         14 . The combination of  claim 9  wherein the article of manufacture is selected from among toy guns, pellet guns, greeting cards, fingerpaints, foot bags, slimy play material, clothing, bubble making toys and bubbles therefor, balloons, bath powders, body lotions, gels, body powders, body creams, toothpastes, mouthwashes, soaps, body paints, bubble bath, board game toys, fishing lures, egg-shaped toys, toy cigarettes, dolls, sparklers, magic wand toys, wrapping paper, gelatins, icings, frostings, fairy dust, beer, ornamental transgenic plants, wine, champagne, milk, soft drinks, ice cubes, ice, dry ice, soaps, body paints and bubble bath.  
     
     
         15 . The combination of  claim 9  that is a transgenic ornamental plant.  
     
     
         16 . The combination of  claim 9  that is a toy.  
     
     
         17 . The combination of  claim 9  that is a food.  
     
     
         18 . The combination of  claim 9  that is a cosmetic.  
     
     
         19 . The combination of  claim 9  that is a beverage.  
     
     
         20 . The combination of  claim 9 , wherein the article of manufacture is selected from among toys, fountains, personal care items, fairy dust, foods, textile, transgenic ornamental plant and paper products.  
     
     
         21 . The combination of  claim 9 , wherein the article of manufacture is selected from among toy guns, pellet guns, greeting cards, fingerpaints, foot bags, slimy play material, clothing, bubble making toys and bubbles therefor, balloons, bath powders, body lotions, gels, body powders, body creams, toothpastes, mouthwashes, soaps, body paints, bubble bath, board game toys, fishing lures, egg-shaped toys, toy cigarettes, dolls, sparklers, magic wand toys, wrapping paper, gelatins, icings, frostings, fairy dust, beer, wine, champagne, soft drinks, ice cubes, ice, dry ice, soaps, body paints and bubble bath.  
     
     
         22 . An antibody that specifically binds to  Renilla reniformis  or a molecule or derivative of the antibody containing the binding domain thereof.  
     
     
         23 . The antibody of  claim 22  that is a monoclonal antibody.  
     
     
         24 . An isolated substantially purified  luciferase  and GFP fusion protein, wherein the GFP is a  Renilla reniformis  GFP and the fusion protein is encoded by a nucleic acid construct comprising a nucleotide sequence encoding a  luciferase  and a nucleotide sequence encoding a  Renilla reniformis  green fluorescent protein, comprising a nucleic acid sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto that encodes a  Renilla reniformis  fluorescent protein (GFP), wherein the encoded  luciferase  and  Renilla reniformis  GFP comprise a fusion protein.  
     
     
         25 . The fusion protein of  claim 24 , wherein the  luciferase  is a  Renilla luciferase.    
     
     
         26 . The fusion protein of  claim 24 , wherein the  luciferase  is a  Renilla reniformis luciferase.    
     
     
         27 . A composition comprising: 
 a nucleic acid construct comprising a nucleotide sequence encoding a  luciferase  and a nucleotide sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto, that encodes a  Renilla reniformis  fluorescent protein (GFP), wherein the encoded  luciferase  and  Renilla reniformis  GFP comprise a fusion protein.    
     
     
         28 . The composition of  claim 27 , further comprising at least one component of a bioluminescence generating system.  
     
     
         29 . The composition of  claim 28 , wherein the component of the bioluminescence generating system is a  luciferin.    
     
     
         30 . A biosensor, comprising a GFP protein encoded a nucleic acid molecule encoding a  Renilla reniformis  green fluorescent protein, comprising a nucleic acid sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto and a  luciferase.    
     
     
         31 . The biosensor of  claim 30 , wherein the  luciferase  is a  Renilla luciferase.    
     
     
         32 . A biosensor of  claim 30 , further comprising a modulator.  
     
     
         33 . A biosensor, comprising the fusion protein of  claim 24 .  
     
     
         34 . A biosensor of  claim 33 , wherein the GFP and  luciferase  in the fusion protein are not contiguous.  
     
     
         35 . A bioluminescence resonance energy transfer (BRET) system, comprising: 
 (a) a GFP encoded by a nucleic molecule encoding a  Renilla reniformis  green fluorescent protein, comprising a nucleic acid sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto;    (b) a  luciferase  from which the GFP can accept energy when the GFP and  luciferase  associate; and    (c) a  luciferin  or other substrate of the  luciferase.      
     
     
         36 . The BRET system of  claim 35 , further comprising one or more modulators.  
     
     
         37 . The BRET system of  claim 36 , wherein the GFP and  luciferase  are each attached to a different modulator, or each are attached to the same modulator.  
     
     
         38 . The BRET system of  claim 36 , wherein a conformational change in a modulator causes an increase in the proximity of the  luciferase  and GFP.  
     
     
         39 . The BRET system of  claim 36 , wherein a conform additional change in a modulator causes a decrease in the proximity of the  luciferase  and GFP.  
     
     
         40 . The BRET system of  claim 36 , wherein the  luciferase  is  Renilla reniformis luciferase.    
     
     
         41 . A microelectronic device, comprising: 
 a substrate;    a plurality of micro-locations defined on the substrate, wherein each micro-location is for linking a macromolecule;    an independent photodetector integrated at or adjacent to each micro-location and optically coupled to each micro-location, each photodetector being configured to generate a sensed signal responsive to the photons of light emitted at the corresponding micro-location when a light-emitting chemical reaction occurs at that micro-location, each photodetector being independent from the photodetectors optically coupled to the other micro-locations; and    an electronic circuit coupled to each photodetector and configured to read the sensed signal generated by each photodetector and to generate output data signals therefrom that are indicative of the light emitted at each micro-location by the light-emitting chemical reactions, whereby the device detects photons of light emitted by light-emitting chemical reactions, wherein: 
 each micro-location is defined by a portion of the surface; and  
 the micro-locations defined on the substrate each comprise a component of a bioluminescence generating system and a green fluorescent protein comprising a nucleic acid sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto, whereby photons of light are emitted when a reaction takes place at that micro-location.  
   
     
     
         42 . The device of  claim 41 , wherein the micro-locations are provided as an array.  
     
     
         43 . The device of  claim 41 , wherein the bioluminescence generating system comprises a  Renilla luciferase.    
     
     
         44 . The device of  claim 42 , wherein the bioluminescence generating system comprises a  Renilla reniformis luciferase.    
     
     
         45 . A method of detecting and identifying analytes in a biological sample, comprising: 
 providing the microelectronic device of  claim 41;     attaching a macromolecule or plurality of different macromolecules to the surface at each micro-location on the device, wherein macromolecule is specific for binding to selected analyte that may be present in the biological sample;    contacting the sample with the surface of the microelectronic device, whereby any of the selected analytes that are present in the sample bind to the macromolecule attached to the surface at each micro-location;    exposing the surface of the microelectronic device to a second macromolecule or plurality thereof bound to the selected analyte already bound to the first macromolecule at each micro-location, wherein the second macromolecule comprises a component of a bioluminescence generating reaction;    initiating the bioluminescence generating reaction by contacting the surface of the device with the remaining components of the bioluminescence generating reaction, wherein the wavelength of the resulting light is shifted by the  Renilla reniformis  GFP; and    detecting photons of light emitted by the GFP using a photodetector optically coupled to each micro-location, each photodetector generating a sensed signal representative of the bioluminescence generation at the respective micro-location.    
     
     
         46 . A transgenic animal or plant that expresses a  Renilla reniformis  nucleic acid encoding a  Renilla reniformis  green fluorescent protein, comprising a nucleic acid sequence that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a nucleic acid molecule of  Renilla reniformis  having at least 80% sequence identity thereto.  
     
     
         47 . The transgenic animal or plant of  claim 46 , selected from among fish, worms, monkeys, rodents, goats, pigs, cow, sheep, horses, flowering plants, ornamental plants.  
     
     
         48 . The transgenic animal or plant of  claim 46  that is an orchid.