Modified green fluorescent proteins and methods for using same

• US 20070105196A1
• Filed: 10/13/2006
• Published: 05/10/2007
• Est. Priority Date: 11/04/2005
• Status: Active Grant

• Current Assignee: Evrogen Joint Stock Company
• Original Assignee: Evrogen Joint Stock Company
• Inventors: Lukyanov, Sergey
• Granted Patent: US 7,417,131 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

The present invention provides nucleic acid molecules encoding mutant fluorescent proteins as well as proteins encoded by these nucleic acids. In addition, host-cells, stable cell lines and transgenic organisms comprising the above-referenced nucleic acid molecules are provided. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly for labeling of biomolecules, cells, or cell organelles.

Claims

1. An isolated nucleic acid, comprising a nucleic acid sequence encoding a genetically engineered functional fluorescent protein substantially similar to the wild-type Aequorea macrodactyla fluorescent protein of SEQ ID NO:

2 and whose amino acid sequence differs from the amino acid sequence of SEQ ID NO;

2 by at least an F220L amino acid substitution, wherein said genetically engineered functional fluorescent protein has an increased maturation speed at a temperature of 20°

C. or above compared to the wild-type Aequorea macrodactyla green fluorescent protein.

2. The nucleic acid of claim 1, wherein the amino acid sequence of the genetically engineered functional fluorescent protein further comprises one or more amino acid substitutions selected from the group consisting of K3G, E6D, T9A, P58T, F99L, F99H, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R.

3. The nucleic acid of claim 2, wherein the nucleic acid sequence encodes a genetically engineered functional fluorescent protein that is substantially identical to the amino acid sequence of SEQ ID NO:

18.

4. The nucleic acid of claim 3, wherein the genetically engineered functional fluorescent protein has an amino acid sequence that is selected from the group consisting of SEQ ID NOS:

18, 20, 22, and 24.

5. A vector comprising the nucleic acid of claim 1.

6. A vector comprising the nucleic acid of claim 2.

7. A vector comprising the nucleic acid of claim 3.

8. A vector comprising the nucleic acid of claim 4.

9. An expression cassette comprising:

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

(b) the nucleic acid according to claim 1;

and

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

10. An expression cassette comprising:

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

(b) the nucleic acid according to claim 2;

and

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

11. An expression cassette comprising:

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

(b) the nucleic acid according to claim 3;

and

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

12. An expression cassette comprising:

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

(b) the nucleic acid according to claim 4;

and

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

13. An host cell or progeny thereof, comprising the expression cassette according to claim 9 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.

14. An host cell or progeny thereof, comprising the expression cassette according to claim 10 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.

15. An host cell or progeny thereof, comprising the 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.

16. An host cell or progeny thereof, comprising the expression cassette according to claim 12 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.

17. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 1.

18. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 2.

19. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 3.

20. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 4.

25. A kit comprising at least one nucleic acid according to claim 1.

21. A genetically engineered functional fluorescent protein whose amino acid sequence is substantially similar to the wild type Aequorea macrodactyla fluorescent protein of SEQ ID NO:

2 and differs from the amino acid sequence of SEQ ID NO;

2 by at least an F220L amino acid substitution, wherein said genetically engineered functional fluorescent protein has an increased maturation speed at a temperature of 20°

C. or above compared to the wild type Aequorea macrodactyla green fluorescent protein.

22. The genetically engineered functional fluorescent protein of claim 21, wherein the genetically engineered functional fluorescent protein comprises one or more amino acid substitutions selected from the group consisting of K3G, E6D, T9A, P58T, F99L, F99H, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R.

23. The genetically engineered functional fluorescent protein of claim 22, wherein the genetically engineered functional fluorescent protein is substantially identical to the amino acid sequence of SEQ ID NO:

18.

24. The genetically engineered functional fluorescent protein of claim 23, wherein the genetically engineered functional fluorescent protein has an amino acid sequence that is selected from the group consisting of SEQ ID NOS:

18, 20, 22, and 24.