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

US 7,109,315 B2
Filed: 03/15/2001
Issued: 09/19/2006
Est. Priority Date: 03/15/2000
Status: Expired due to Term

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1. An isolated nucleic acid molecule encoding a Renilla reniformis green fluorescent protein, comprising 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.

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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.

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29 Claims

1. An isolated nucleic acid molecule encoding a Renilla reniformis green fluorescent protein, comprising 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.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
- - 2. An isolated nucleic acid molecule of claim 1 that encodes a protein having at least 90% sequence identity to the protein of SEQ ID NO. 27.
  - 3. The isolated nucleic acid molecule of claim 1, comprising a nucleotide sequence selected from the group consisting of:
    - (a) the coding portion of the nucleotide sequence set forth in any of SEQ ID NOs. 23-25;
      
      (b) a nucleotide sequence that hybridizes under conditions of 0.1×
      
      standard saline phosphate EDTA buffer, 0.1% SDS at 65°
      
      C. to the nucleotide sequence of (a); and
      
      (c) a nucleotide sequence comprising degenerate codons of (a) or (b).
  - 4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid is DNA.
  - 5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid is RNA.
  - 6. A nucleic acid probe or primer, comprising at least 14 contiguous nucleotides selected from the nucleotide sequence set of claim 1.
  - 7. The probe or primer of claim 6, comprising at least 16 contiguous nucleotides selected from the nucleotide sequence in claim 1.
  - 8. The probe or primer of claim 7, comprising at least 30 contiguous nucleotides.
  - 9. A plasmid, comprising the nucleotide sequence of claim 1.
  - 10. A cell comprising the plasmid of claim 9, 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 an animal cell.
  - 11. The plasmid of claim 9 that is an expression vector, further comprising:
    - a promoter element;
      
      a cloning site for the introduction of the nucleic acid molecule; and
      
      a marker gene;
      
      wherein the nucleic acid comprising the cloning site is positioned between nucleic acids encoding the promoter element and the green fluorescent protein and wherein the nucleic acid encoding the green fluorescent protein is operatively linked to the promoter element.
  - 12. The plasmid of claim 11, wherein the marker gene comprises a nucleotide sequence encoding a luciferase.
  - 13. A recombinant host cell, comprising the plasmid of claim 9.
  - 14. The cell of claim 13, 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 an animal cell.
  - 15. A reporter gene construct, comprising the nucleic acid molecule of claim 1.
  - 16. A nucleic acid construct, comprising the nucleotide sequence encoding a luciferase and a nucleotide sequence of claim 1 that encodes a Renilla reniformis fluorescent protein (GFP).
  - 17. The nucleic acid construct of claim 16, wherein the luciferase is a Renilla mulleri luciferase, a Gaussia luciferase or a Pleuromamma luciferase.
  - 18. The nucleic acid construct of claim 17, wherein the Gaussia luciferase is a Gaussia princeps luciferase.
  - 19. The nucleic acid construct of claim 16, wherein the luciferase is encoded by:
    - a nucleotide sequence set forth in SEQ ID NO. 17, SEQ ID NO. 19, or SEQ ID NO. 28;
      
      a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO. 18, SEQ ID NO. 20 or SEQ ID NO. 29; and
      
      a nucleotide sequence that hybridizes under the condition of claim 3 to the nucleotide sequence set forth in SEQ ID NO. 17, SEQ ID NO. 19 or SEQ ID NO. 28.
  - 20. The nucleic acid construct of claim 16, that is DNA.
  - 21. The nucleic acid construct of claim 16, that is RNA.
  - 22. A plasmid, comprising the nucleic acid construct of claim 16.
  - 23. The plasmid of claim 22, further comprising a nucleotide sequence encoding:
    - a promoter element; and
      
      a selectable marker;
      
      wherein, the nucleotide sequence encoding the luciferase and Renilla reniformis GFP is operatively linked to the promoter element, whereby the luciferase and Renilla reniformis GFP are expressed.
  - 24. The construct of claim 16, wherein the luciferase and the GFP are encoded by a polycistronic message.
  - 25. The construct of claim 16, wherein the encoded luciferase and Renilla reniformis GFP comprise a fusion protein.
  - 26. The nucleic acid construct of claim 25, wherein the nucleotide sequence that encoding the luciferase and GFP are not contiguous.
  - 27. The nucleic acid construct of claim 26, comprising a nucleotide sequence that encodes a ligand binding domain of a target protein.
  - 28. The construct of claim 16, wherein the luciferase is Renilla reniformis luciferase.
  - 29. A recombinant host cell, comprising the plasmid of claim 22.

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Current Assignee
Gaussia, LLC, Prolume Limited
Original Assignee
Bruce J. Bryan, Prolone Limited
Inventors
Szent-Gyorgyi, Christopher, Szczepaniak, William, Bryan, Bruce
Primary Examiner(s)
Carlson, Karen Cochrane
Assistant Examiner(s)
Liu, Samuel W.

Application Number

US09/808,898
Publication Number

US 20030092098A1
Time in Patent Office

2,014 Days
Field of Search

536/23.1, 435/69.1, 435/325, 435/252.3, 435/320.1
US Class Current

536/23.1
CPC Class Codes

C07K 14/43595   from coelenteratae, e.g. me...

C07K 2319/00   Fusion polypeptide

C12N 9/0069   acting on single donors wit...

C12Y 113/12005   Renilla-luciferin 2-monooxy...

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

First Claim

6 Assignments

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Abstract

175 Citations

29 Claims

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

First Claim

6 Assignments

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Litigations

0 Petitions

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Accused Products

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Abstract

175 Citations

29 Claims

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