Rapidly maturing fluorescent proteins and methods for using the same

• US 8,664,471 B2
• Filed: 05/11/2004
• Issued: 03/04/2014
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Chicago, University of Washington
• Inventors: Bevis, Brooke, Glick, Benjamin
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US10/844,064
• Publication Number:
  US 20050149994A1
• Time in Patent Office: 3,584 Days
• Field of Search: None
• US Class Current: 800/13
• 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 polynucleotide encoding a fluorescent mutant of wild-type DsRed comprising an amino acid substitution at amino acid position 42 of SEQ ID NO:

2, wherein the mutant matures more rapidly than the wild-type DsRed.

2. The polynucleotide of claim 1, wherein the mutant further comprises a mutation in at least one amino acid position selected from the group consisting of amino acid residues 2, 5, 6, 21, 41, 44, 117, 145, and 217 of SEQ ID NO:

2.

3. The polynucleotide of claim 1, wherein the mutant comprises the mutation N42Q.

4. The polynucleotide of claim 1, wherein the mutant comprises the mutation N42H.

5. The polynucleotide of claim 2, wherein the mutant comprises a mutation at amino acid position 41 of SEQ ID NO:

2.

6. The polynucleotide of claim 5, wherein the mutation at amino acid position 41 of SEQ ID NO:

2 is selected from the group consisting of H41T and H41L.

7. The polynucleotide of claim 5, wherein the mutant further comprises a mutation at amino acid position 44 of SEQ ID NO:

2.

8. The polynucleotide of claim 7, wherein the mutation at amino acid position 44 of SEQ ID NO:

2 is V44A.

9. The polynucleotide of claim 2, wherein the mutant comprises a mutation at amino acid position 44 of SEQ ID NO:

2.

10. The polynucleotide of claim 9, wherein the mutation at amino acid position 44 of SEQ ID NO:

2 is V44A.

11. The polynucleotide of claim 1, wherein the mutant comprises mutations H41T, N42Q, and V44A of SEQ ID NO:

2.

12. The polynucleotide of claim 2, wherein the mutant further comprises mutation T21S.

13. The polynucleotide of claim 2, wherein the mutant further comprises a mutation that results in a reduction in the net charge near the N terminus.

14. The polynucleotide of claim 13, wherein the mutation that results in a reduction in the net charge near the N terminus is selected from the group consisting of R2H, R2L, R2A, K5E, K5Q, K5M, and N6D.

15. The polynucleotide of claim 14, wherein the mutant comprises mutations R2A, K5E, and N6D.

16. The polynucleotide of claim 15, further comprising mutations T21S, H41T, N42Q and V44A.

17. The polynucleotide of claim 16, further comprising mutations C117S and T217A.

18. The polynucleotide of claim 16, further comprising mutation A145P.

19. The polynucleotide of claim 18, further comprising mutation T217A.

20. The polynucleotide of claim 2, wherein the mutant comprises mutation T217A.

21. The polynucleotide of claim 2, wherein the mutant comprises a mutation selected from the group consisting of A145P and A145S.

22. The polynucleotide of claim 2, wherein the mutant comprises a mutation selected from the group consisting of C117S and C117A.

23. A polynucleotide comprising the polynucleotide of claim 1 operably linked to a promoter.

24. A cell comprising the polynucleotide of claim 1.

25. A method of producing a fluorescent polypeptide comprising growing the cell of claim 24, whereby the polynucleotide is expressed to produce the fluorescent polypeptide.

26. A non-human transgenic organism comprising the polynucleotide of claim 1.

27. A kit comprising the polynucleotide of claim 1.

28. The polynucleotide of claim 1, wherein the fluorescent mutant further comprises amino acid substitutions at amino acid positions 2, 5, 6, 21, 41 and 44 of SEQ ID NO:

2.

29. The polynucleotide of claim 1, wherein the fluorescent mutant is a fusion protein.

30. A method of detecting expression of a fusion protein, comprising obtaining expression of the polynucleotide of claim 29 and detecting fluorescence.