Mutant luciferase of a firefly, mutant luciferase genes, recombinant DNAS containing the genes and a method of producing mutant luciferase

• US 5,219,737 A
• Filed: 03/26/1991
• Issued: 06/15/1993
• Est. Priority Date: 03/27/1990
• Status: Expired due to Fees

• Current Assignee: Kikkoman Corporation
• Original Assignee: Kikkoman Corporation
• Inventors: Kajiyama, Naoki, Nakano, Eiichi
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): PROUTY, REBECCA E
• Application Number:
  US07/675,211
• Time in Patent Office: 812 Days
• Field of Search: 435/8, 435/69.1, 435/71.1, 435/71.2, 435/172.1, 435/172.3, 435/189, 935/10, 935/14, 935/27, 935/56, 536/27
• US Class Current: 435/189
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase

Abstract

The present invention provides industrially useful luciferase. Mutant luciferase of the invention is produced by culturing a microorganism belonging to the genus Escherichia which harbors a recombinant DNA containing the mutant luciferase gene of a firefly. Mutant luciferase can produce red, orange or green color of light which can not be produced by wild type luciferase. Mutant luciferase can be used to measure ATP accurately in a colored solution such as red (e.g., blood), orange, or green in which wild-type luciferase has not provided reliable results.

Claims

1. A mutant luciferase gene encoding the amino acid sequence of luciferase of Luciola cruciata, in which one of the following changes appears:

serine is replaced by asparagine at amino acid 286, glycine is replaced by serine at amino acid 326, histidine is replaced by tyrosine at amino acid 433 or proline is replaced by serine at amino acid 452.

2. The mutant luciferase gene according to claim 1, in which serine is replaced by asparagine at amino acid 286.

3. The mutant luciferase gene according to claim 1, in which glycine is replaced by serine at amino acid 326.

4. The mutant luciferase gene according to claim 1, in which histidine is replaced by tyrosine at amino acid 433.

5. The mutant luciferase gene according to claim 1, in which proline is replaced by serine at amino acid 452.

6. A recombinant DNA comprising the mutant luciferase gene of claim 1.

7. A recombinant DNA comprising the mutant luciferase gene of claim 2.

8. A recombinant DNA comprising the mutant luciferase gene of claim 3.

9. A recombinant DNA comprising the mutant luciferase gene of claim 4.

10. A recombinant DNA comprising the mutant luciferase gene of claim 5.

11. A method of producing a mutant firefly luciferase, which comprises culturing, in a culture medium, a microorganism belonging to the genus Escherichia transformed with a recombinant DNA containing a mutant gene encoding the amino acid sequence of luciferase of Luciola criciata, in which one of the following changes appears:

serine is replaced by asparagine at amino acid 286, glycine is replaced by serine at amino acid 326, histidine is replaced by tyrosine at amino acid 433 or proline is erplaced by serine at amino acid 452, and recovering the mutant luciferase from the culture.

12. The method according to claim 11, in which the microorganism contains recombinant DNA having the mutant gene encoding the amino acid sequence of Luciola cruciata luciferase in which serine is replaced by asparagine at amino acid 286.

13. The method according to claim 11, in which the microorganism contains recombinant DNA having the mutant gene encoding the amino acid sequence of Luciola cruciata luciferase in which glycine is replaced by serine at amino acid 326.

14. The method according to claim 11, in which the microorganism contains recombinant DNA having the mutant gene encoding the amino acid sequence of Luciola cruciata luciferase in which histidine is replaced by tyrosine at amino acid 433.

15. The method according to claim 11, in which the microorganism contains recombinant DNA having the mutant gene encoding the amino acid sequence of Luciola cruciata luciferase in which proline is replaced by serine at amino acid 452.