Renilla reniformis fluorescent proteins, nucleic acids encoding the fluorescent 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 nucleic acid molecule comprising a sequence of nucleotides that encodes a protein of any of SEQ ID NOS. 15, 16, 17, 18, 19, 20, 30 and/or 31, or a protein encoded by a  Renilla mulleri, Ptilosarcus  or  Gaussia  having at least 80% nucleotide sequence identity thereto.  
     
     
         2 . The isolated substantially purified nucleic acid molecule of  claim 1 , wherein the encoded protein is a green fluorescent protein.  
     
     
         3 . The isolated substantially purified nucleic acid molecule of  claim 1 , that encodes a protein having at least 90% sequence identity to the protein of any of SEQ ID NOS. 15, 16, 17, 18, 19, 20, 30 and/or 31.  
     
     
         4 . The isolated substantially purified nucleic acid molecule of  claim 1 , comprising a sequence of nucleotides selected from the group consisting of: 
 (a) the coding portion of the sequence of nucleotides set forth in any of SEQ ID NOS. 15, 16, 17, 18, 19, 20, 30 and/or 31;    (b) a sequence of nucleotides that hybridizes under high stringency to the sequence of nucleotides of (a); and    (c) a sequence of nucleotides comprising degenerate codons of (a) or (b).    
     
     
         5 . The isolated substantially purified nucleic acid molecule of  claim 1 , wherein the nucleic acid is DNA.  
     
     
         6 . The isolated substantially purified nucleic acid molecule of  claim 1 , wherein the nucleic acid is RNA.  
     
     
         7 . A nucleic acid probe or primer, comprising at least 14 contiguous nucleotides selected from any of the sequences of nucleotides of  claim 1 .  
     
     
         8 . The nucleic acid probe or primer of  claim 7 , comprising at least 16 contiguous nucleotides selected from any of the sequences of nucleotides of  claim 1 .  
     
     
         9 . The nucleic acid probe or primer of  claim 8 , comprising at least 30 contiguous nucleotides selected from any of the sequences of nucleotides of  claim 1 .  
     
     
         10 . A plasmid, comprising any of the sequences of nucleotides of  claim 1 .  
     
     
         11 . A recombinant host cell, comprising the plasmid of  claim 10 .  
     
     
         12 . The recombinant host cell of  claim 11 , wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a plant cell, an insect cell, an animal cell or a human cell.  
     
     
         13 . An isolated substantially purified  Renilla mulleri  or  Ptilosarcus  green fluorescent protein encoded by the nucleic acid molecule of  claim 1 .  
     
     
         14 . A composition, comprising the green fluorescent protein of  claim 13  and at least one component of a bioluminescence generating system.  
     
     
         15 . The composition of  claim 14 , 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 crustacea system, a fish system, an annelid system, and an earthworm system.  
     
     
         16 . The composition of  claim 15 , 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,  such  Pachystomias, Malacosteus, Gonadostomias, Gaussia, Watensia, Halisturia,  Vampire squid,  Glyphus, Mycotophids, Vinciguerria, Howella, Florenciella, Chaudiodus, Melanocostus,  Sea Pens,  Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia,  cuttlefish,  Sepiolina, Oplophorus, Acanthophyra, Sergestes, Gnathophausia, Argyropelecus, Yarella, Diaphus, Gonadostomias  and  Neoscopelus.    
     
     
         17 . A reporter gene construct, comprising the nucleic acid molecule of  claim 1 .  
     
     
         18 . A combination, comprising an article of manufacture, and a green fluorescent protein encoded by the nucleic acid molecule of  claim 1 .  
     
     
         19 . The combination of  claim 18 , further comprising at least one component of a bioluminescence generating system, whereby the combination is a novelty item.  
     
     
         20 . The combination of  claim 18 , wherein the component of the bioluminescence generating system comprises a  luciferase  and/or a  luciferin.    
     
     
         21 . The combination of  claim 18 , wherein the article of manufacture is selected from among toys, fountains, personal care items, cosmetics, fairy dust, foods and beverages, textile and paper products, 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.  
     
     
         22 . An antibody or a molecule or derivative of the antibody containing the binding domain thereof that specifically binds to the nucleic acid molecule or a protein encoded by a  Renilla mulleri  or  Ptilosarcus  of  claim 1 .  
     
     
         23 . The antibody of  claim 22  that is a monoclonal antibody.  
     
     
         24 . A nucleic acid construct, comprising a nucleotide sequence encoding a  luciferase  and a sequence of nucleotides of  claim 1  that encodes a  Renilla mulleri  or  Ptilosarcus  green fluorescent protein.  
     
     
         25 . The nucleic acid construct of  claim 24 , wherein the  luciferase  is encoded by: 
 a sequence of nucleotides set forth in SEQ ID No. 17, SEQ ID No.19, or SEQ ID No. 28;    a sequence of nucleotides encoding the amino acid sequence set forth in SEQ ID No.18, SEQ ID No. 20 or SEQ ID No.29; and    a sequence of nucleotides that hybridizes under high stringency to the sequence of nucleotides set forth in SEQ ID No.17, SEQ ID No. 19 or SEQ ID No.28.    
     
     
         26 . The nucleic acid construct of  claim 24 , that is DNA.  
     
     
         27 . The nucleic acid construct of  claim 24 , that is RNA.  
     
     
         28 . A plasmid, comprising the nucleic acid construct of  claim 24 .  
     
     
         29 . A recombinant host cell, comprising the plasmid of  claim 24 .  
     
     
         30 . The recombinant host cell of  claim 29 , wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a plant cell, an insect cell, and animal cell and a human cell.  
     
     
         31 . An isolated substantially purified  luciferase  and green fluorescent protein fusion protein, wherein the green fluorescent protein is a  Renilla mulleri  or  Ptilosarcus  green fluorescent protein and the fusion protein is encoded by the nucleic acid construct of  claim 24 .  
     
     
         32 . A biosensor, comprising a green fluorescent protein encoded by the nucleic acid molecule of  claim 1  and a  luciferase.    
     
     
         33 . A biosensor of claim  59 , further comprising a modulator.  
     
     
         34 . A biosensor, comprising a fusion protein of  claim 31 .  
     
     
         35 . A bioluminescence resonance energy transfer (BRET) system, comprising: (a) a GFP encoded by the nucleic molecule of  claim 1;  (b) a  luciferase  from which the GFP can accept energy; (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 35 , 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 35 , wherein a conformation change in a modulator causes an increase in the proximity of the  luciferase  and GFP.  
     
     
         39 . The BRET system of  claim 35 , wherein a conformational change in a modulator causes a decrease in the proximity of the  luciferase  and GFP.  
     
     
         40 . The BRET system of  claim 35 , wherein the  luciferase  is  Renilla mulleri luciferase.    
     
     
         41 . A transgenic animal or plant that expresses the nucleic acid of  claim 1 .  
     
     
         42 . The transgenic animal or plant of  claim 41 , selected from among fish, worms, monkeys, rodents, goats, pigs, cows, sheep, horses, flowering plants, ornamental plants.  
     
     
         43 . The nucleic acid molecule of  claim 1 , that is optimized for expression in plant cells, yeast cells, bacterial cells, fungal cells, insect cells or animal cells.  
     
     
         44 . The nucleic acid molecule of  claim 43 , that is optimized for expression in human cells.  
     
     
         45 . An isolated substantially purified nucleic acid molecule encoding a green fluorescent protein, comprising a sequence of nucleotides that encodes the protein of SEQ ID NO. 27 or a green fluorescent protein encoded by a  Renilla reniformis  having at least 80% nucleotide sequence identity thereto that is optimized for expression in plant cells, yeast cells, bacterial cells, fungal cells, insect cells or animal cells.  
     
     
         46 . The nucleic acid molecule of  claim 45 , that is optimized for expression in human cells.