Novel Branchiostoma Derived Fluorescent Proteins

• US 20100168396A1
• Filed: 09/24/2007
• Published: 07/01/2010
• Est. Priority Date: 09/22/2006
• Status: Active Grant

• Current Assignee: Stowers Institute for Medical Research
• Original Assignee: Stowers Institute for Medical Research
• Inventors: Cook, Malcolm, Schwartz, Joel, Mushegian, Arcady, Ma, Limei, Yu, Congrong (Ron)
• Granted Patent: US 8,680,235 B2
• Time in Patent Office: Days
• US Class Current: 530/387.900
• CPC Class Codes: C07K 14/43504 from invertebrates

Abstract

The present invention provides compositions, combinations, methods, sequences and kits for use of novel fluorescent proteins derived from the genus Branchiostoma. Specifically, polynucleotide and polypeptide sequences encoding fluorescent proteins isolated from Branchiostoma floridae, including harmonized sequences, which permit enhanced expression of the encoded polypeptides in mammalian cells in vivo are provided.

Claims

1. An isolated Branchiostoma fluorescent protein, characterized by having a cellular fluorescence that is at least about five times greater than the cellular fluorescence of a GFP protein that fluoresces at normal conditions in cells, and having a chromophore region comprising amino acid sequence GYG.

2. The isolated Branchiostoma fluorescent protein of claim 1 having a chromophore region consisting of amino acid sequence LGYG (SEQ ID NO:

36).

3. The isolated Branchiostoma fluorescent protein of claim 1, which has a polypeptide sequence that has at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31 and SEQ ID NO;

38.

6. A fusion protein comprising a protein of interest operably linked to the isolated Branchiostoma fluorescent protein of claim 1.

7. The fusion protein of claim 6, wherein the protein of interest is operably linked by a linker sequence selected from the group consisting of (Arg-Ala-Arg-Asp-Pro-Arg-Val-Pro-Val-Ala-Thr)1-5 (SEQ ID NO:

32), (Gly-Ser)1-15, (Thr-Ser-Pro)1-15, (Gly-Gly-Gly)1-15, (Glu-Lys)1-15, and (Gly4Ser)2-4.

8. A composition comprising an antibody, wherein the antibody binds substantially to the isolated Branchiostoma fluorescent protein of claim 1.

9. The composition of claim 8, wherein the antibody is operatively linked to at least a first therapeutic or diagnostic agent.

10. A method of making the Branchiostoma fluorescent protein of claim 1 comprising transfecting a cell with a nucleic acid sequence at least about 90% identical to a polynucleotide selected from the group consisting of SEQ ID NOs:

22-27, 37, and their respective complementary sequences.

11. A kit comprising the isolated Branchiostoma fluorescent protein of claim 1.

12. A kit comprising the antibody of claim 8.

4. An isolated Branchiostoma fluorescent protein, which has a polypeptide sequence that is selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

38, and proteins having a polypeptide sequence that is at least 70% identity to the listed sequences.

5. The isolated Branchiostoma fluorescent protein of claim 4, wherein an amino acid substitution occurs at one of amino acid positions 44, 57, 58, 59, 60, 61, 147, 157, 167, 177, or 201 in the listed sequences.

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. An isolated fluorescent polypeptide, the amino acid sequence of which comprises a sequence selected from the group consisting of SEQ ID NO:

1 and sequences having at least 95% identity to SEQ ID NO;

1.

22. The isolated fluorescent polypeptide of claim 21, the amino acid sequence of which comprises SEQ ID NO:

1.

23. The isolated fluorescent polypeptide of claim 22, the amino acid sequence of which consists of SEQ ID NO:

1.

27. The isolated fluorescent polypeptide of any one of claim 21 or 24, wherein an amino acid substitution occurs at one of amino acid positions 44, 57, 58, 59, 60, 61, 147, 157, 167, 177, or 201 of SEQ ID NO:

1 or SEQ ID NO;

2.

24. An isolated fluorescent polypeptide, the amino acid sequence of which comprises a sequence selected from the group consisting of SEQ ID NO:

2 and sequences having at least 95% identity to SEQ ID NO;

2.

25. The isolated fluorescent polypeptide of claim 24, the amino acid sequence of which comprises SEQ ID NO:

2.

26. The isolated fluorescent polypeptide of claim 24, the amino acid sequence of which consists of SEQ ID NO:

2.

28. A fusion protein comprising a first polypeptide operably linked to a fluorescent polypeptide, the amino acid sequence of the fluorescent polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, and sequences having at least 95% identity to SEQ ID NO;

1 or SEQ ID NO;

2.

29. The fusion protein of claim 28, wherein the first polypeptide is operably linked to the fluorescent polypeptide by a linker sequence selected from the group consisting of (Arg-Ala-Arg-Asp-Pro-Arg-Val-Pro-Val-Ala-Thr)1-5 (SEQ ID NO:

32), (Gly-Ser)1-15, (Thr-Ser-Pro)1-15, (Gly-Gly-Gly)1-15, (Glu-Lys)1-15, and (Gly4Ser)2-4.