Modified bioluminescent proteins and their use

• US 5,683,888 A
• Filed: 07/05/1994
• Issued: 11/04/1997
• Est. Priority Date: 07/22/1989
• Status: Expired due to Fees

• Current Assignee: University College Cardiff Consultants Ltd (Cardiff University)
• Original Assignee: University Of Wales College Of Medicine (University of Cardiff)
• Inventors: Campbell, Anthony Keith
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/270,314
• Time in Patent Office: 1,218 Days
• Field of Search: 435/188, 435/189, 435/8, 435/69.1, 435/69.7, 435/7.6, 435/29, 435/4, 530/409, 530/403, 530/350, 530/402, 536/23.2, 536/23.4, 536/23.5, 536/23.7
• US Class Current: 435/8
• CPC Class Codes: A61K 49/0013 Luminescence
• CPC Class Codes: C07K 14/43536 from worms
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• 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: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

A modified bioluminescent protein responds to different physical, chemical, biochemical or biological conditions to produce light or radiation of altered characteristics when the bioluminescent reaction is initiated. The modified bioluminescent protein may respond to modification thereof, e.g. by covalent modification. The protein may include signal peptides to "target" it. DNA coding for the bioluminescent protein may be altered to include tissue specific promoter or enhancer genes so that the altered DNA acts as reporter gene.

Claims

1. A modified bioluminescent protein selected from the group consisting of firefly luciferase, aequorin and obelin, modified to include an interaction site for a selected analyte, said site being not present in the unmodified protein, wherein the analyte interacts with said interaction site, the bioluminescent protein being reactive in a bioluminescent reaction to produce light, wherein the light is produced having a physical property of a first characteristic when the analyte is interacting with the interaction site, and is produced having a second different characteristic of said physical property when the analyte is not interacting with the interaction site;

said interaction site being selected from the group consisting of kemptide covalently bound to the N or C terminus of firefly luciferase, kemptide covalently bound to the N terminus of aequorin, kemptide covalently bound to the N terminus of obelin, malantide covalently bound to the N terminus of aequorin, and an RRXS phosphorylation site at amino acid 217 of firefly luciferase.

2. A bioluminescent protein according to claim 1 for intracellular use, wherein said protein includes a signal peptide for targeting said protein to a specific organelle or site within a cell.

4. Nucleic acid coding for the bioluminescent protein as claimed in claim 1.

5. Nucleic acid according to claim 4, including promoter or enhancer sequences specific to a tissue or substance of interest.

6. Nucleic acid according to claim 4, including a nucleotide sequence coding for an enzyme for producing luciferin.

7. A method of detecting a change in a physical, chemical, biochemical or biological condition, the method comprising:

providing a bioluminescent protein according to claim 1 in a reaction system containing an analyte of interest;

observing whether light of the first and/or second characteristic is produced; and

detecting said change based on production of light of said first and/or second characteristic.

8. A method of detecting, locating or measuring a substance of biological interest, the method comprising:

(a) providing a bioluminescent protein according to claim 1 in a reaction system containing an analyte of interest;

(b) analyzing said first and/or second characteristics of the light produced; and

(c) detecting, locating or measuring said analyte based on said first and/or second characteristic of the light produced.

9. A method of detecting, locating, measuring a substance of biological interest within a cell which comprises:

introducing into said cell nucleic acid coding for a bioluminescent protein according to claim 1,

causing said modified bioluminescent protein to be expressed,

causing said bioluminescent protein to take part in a bioluminescent reaction,

analyzing said first and/or second characteristics of the light produced; and

detecting, locating or measuring said analyte based on said first and/or second characteristic of the light produced.

10. A method according to claim 9, wherein said nucleic acid includes a promoter or enhancer sequence specific to a substance of interest.

3. A modified bioluminescent protein selected from the group consisting of firefly luciferase, aequorin and obelin, modified to include an interaction site for a substance of interest, said site being not present in the unmodified protein and reactive in a bioluminescent reaction, wherein light emitted in said bioluminescent reaction involving said protein changes depending on the concentration of said substance of interest;

said interaction site being selected from the group consisting of kemptide covalently bound to the N or C terminus of firefly luciferase, kemptide covalently bound to the N terminus of aequorin, kemptide covalently bound to the N terminus of obelin, malantide covalently bound to the N terminus of aequorin, and an RRXS phosphorylation site at amino acid 217 of firefly luciferase.