Rapidly maturing fluorescent proteins and methods for using the same

• US 20050149994A1
• Filed: 05/11/2004
• Published: 07/07/2005
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Chicago
• Inventors: Bevis, Brooke, Glick, Benjamin
• Granted Patent: US 8,664,471 B2
• Time in Patent Office: Days
• US Class Current: 800/3
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Nucleic acid compositions encoding rapidly maturing fluorescent proteins, as well as non-aggregating versions thereof (and mutants thereof) as well as the proteins encoding the same, are provided. The proteins of interest are proteins that are fluorescent, where this feature arises from the interaction of two or more residues of the protein. The subject proteins are further characterized in that, in certain embodiments, they are mutants of wild type proteins that are obtained either from non-bioluminescent Cnidarian, e.g., Anthozoan, species or are obtained from Anthozoan non-Pennatulacean (sea pen) species. In certain embodiments, the subject proteins are mutants of wild type Discosoma sp. “red” fluorescent protein. Also of interest are proteins that are substantially similar to, or mutants of, the above specific proteins. Also provided are fragments of the nucleic acids and the peptides encoded thereby, as well as antibodies to the subject proteins and transgenic cells and organisms. The subject protein and nucleic acid compositions find use in a variety of different applications. Finally, kits for use in such applications, e.g., that include the subject nucleic acid compositions, are provided.

Claims

1. A nucleic acid that encodes a rapidly maturing chromo- or fluorescent mutant of a Cnidarian chromo- or fluorescent protein or mutant thereof.

2. The nucleic acid according to claim 1, wherein said Cnidarian chromo- or fluorescent protein is from a non-bioluminescent Cnidarian species.

3. The nucleic acid according to claim 2, wherein said non-bioluminescent Cnidarian species is an Anthozoan species.

4. The nucleic acid according to claim 3, wherein said nucleic acid Anthozoan species is Discosoma.

5. The nucleic acid according to claim 4, wherein said nucleic acid encodes a mutant of DsRed.

6. The nucleic acid according to claim 5, wherein said nucleic acid encodes. a product having a point mutation at at least one of position 2, position 5, position 6, position 21, position 41, position 42, position 44, and position 117 relative to wild type DsRed.

7. The nucleic acid according to claim 6, wherein said product is a product having a point mutation at at least one of position 145 and 217.

8. A nucleic acid according to claim 7, wherein said nucleic acid has a sequence of residues that is substantially the same as or identical to a nucleotide sequence of at least 10 residues in length of SEQ ID NOS:

01 or 02.

9. A fragment of the nucleic acid selected according to claim 1.

10. A construct comprising a vector and a nucleic acid according to claim 1.

11. An expression cassette comprising:

(a) a transcriptional initiation region functional in an expression host;

(b) a nucleic acid according to claim 1;

and

(c) and a transcriptional termination region functional in said expression host.

12. A cell, or the progeny thereof, comprising an expression cassette according to claim 11 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell.

13. A method of producing a chromo and/or fluorescent protein, said method comprising:

growing a cell according to claim 12, whereby said protein is expressed; and

isolating said protein substantially free of other proteins.

14. A protein or fragment thereof encoded by a nucleic acid according to claim 1.

15. An antibody binding specifically to a protein according to claim 14.

16. A transgenic cell or the progeny thereof comprising a transgene that is a nucleic acid according to claim 1.

17. A transgenic organism comprising a transgene that is a nucleic acid according to claim 1.

18. In an application that employs a chromo- or fluorescent protein, the improvement comprising:

employing a protein according to claim 14.

19. In an application that employs a nucleic acid encoding a chromo- or fluorescent protein, the improvement comprising:

employing a nucleic acid according to claim 1.

20. A kit comprising a nucleic acid according to claim 1.