Thermostable luciferase of firefly, thermostable luciferase gene of firefly, novel recombinant DNA, and process for the preparation of thermostable luciferase of firefly

• US 5,229,285 A
• Filed: 06/23/1992
• Issued: 07/20/1993
• Est. Priority Date: 06/27/1991
• Status: Expired due to Term

• 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:
  US07/903,047
• Time in Patent Office: 392 Days
• Field of Search: 435/69.1, 435/71.2, 435/252.3, 435/252.33, 435/320.1, 435/172.3, 435/172.1, 536/23.2, 935/9, 935/14, 935/29, 935/56, 935/72, 935/73, 935/10
• 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: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase

Abstract

The present invention relates to thermostable luciferase of firefly wherein an amino acid at the 217-position of the amino acid sequence of wild-type firefly luciferase or an amino acid equivalent to the amino acid at the 217-position of luciferase of GENJI firefly or HEIKE firefly is converted into a hydrophobic amino acid, a gene encoding said thermostable luciferase, a vector comprising the gene encoding said thermostable luciferase inserted therein, and a process for the preparation of thermostable firefly luciferase comprising use of said vector.

Claims

1. A gene encoding a thermostable luciferase of firefly, which encodes the amino acid sequence of the wild-type firefly luciferase of Luciola cruciata (Genji firefly, Seq. ID No. 2) or Luciola lateralis (Heike firefly, Seq. ID No. 8), in which the amino acid at the 217-position is replaced by a hydrophobic amino acid.

2. A gene encoding a thermostable luciferase of firefly according to claim 1, wherein the hydrophobic amino acid is isoleucine, leucine, or valine.

3. Recombinant DNA, which comprises the gene encoding a thermostable luciferase of firefly according to claim 1 inserted into vector DNA.

4. Process for the preparation of a thermostable luciferase of firefly, which comprises incubation in a culture medium of a microorganism belonging to the genus Escherichia, carrying the recombinant DNA according to claim 3 and being capable of producing thermostable luciferase of firefly, and subsequent collection of thermostable luciferase of firefly from said culture medium.

5. A thermostable luciferase of firefly, wherein an amino acid at the 217-position of the amino acid sequence of wild-type firefly luciferase of Luciola cruciata (GENJI SEQ. ID No. 2firefly) or Luciola lateralis (HEIKE firefly SEQ. ID. No. 8) is replaced by a hydrophobic amino acid.