Branchiostoma derived fluorescent proteins

• US 8,680,235 B2
• Filed: 09/24/2007
• Issued: 03/25/2014
• Est. Priority Date: 09/22/2006
• Status: Active Grant

• Current Assignee: Stowers Institute for Medical Research
• Original Assignee: Stowers Institute for Medical Research
• Inventors: Mushegian, Arcady, Yu, Congrong (Ron), Schwartz, Joel, Ma, Limei, Cook, Malcolm
• Primary Examiner(s): Minnifield, Nita M
• Application Number:
  US12/311,076
• Publication Number:
  US 20100168396A1
• Time in Patent Office: 2,374 Days
• Field of Search: None
• US Class Current: 530/350
• 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, which has a polypeptide sequence that is selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, a polypeptide sequence that has at least 90% identity to SEQ ID NO;

1, and a polypeptide sequence that has at least 90% identity to SEQ ID NO;

2.

2. The isolated Branchiostoma fluorescent protein of claim 1, 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.

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

4. The fusion protein of claim 3, 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.

5. A method of making the Branchiostoma fluorescent protein of claim 1 comprising

(i) 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:

22-23, 25-26, and 37;

(ii) growing the cell expressing the polypeptide; and

(iii) isolating the polypeptide.

6. A kit comprising the isolated Branchiostoma fluorescent protein of claim 1 and a buffered solution.

7. 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.

8. The isolated fluorescent polypeptide of claim 7, the amino acid sequence of which comprises SEQ ID NO:

1.

9. The isolated fluorescent polypeptide of claim 8, the amino acid sequence of which consists of SEQ ID NO:

1.

17. A kit comprising the isolated Branchiostoma fluorescent protein of claim 7 and a buffered solution.

10. 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.

11. The isolated fluorescent polypeptide of claim 10, the amino acid sequence of which comprises SEQ ID NO:

2.

12. The isolated fluorescent polypeptide of claim 10, the amino acid sequence of which consists of SEQ ID NO:

2.

13. The isolated fluorescent polypeptide of claim 7 or 10, 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.

16. A kit comprising the isolated Branchiostoma fluorescent protein of claim 10 and a buffered solution.

14. 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.

15. The fusion protein of claim 14, 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.