Calcium-binding photoprotein, gene encoding the same, and use thereof

1. A method for detecting calcium ions in a solution, comprising the following steps;

Step (1): providing a holoprotein comprising a peroxide of coelenterazine or a peroxide of a coelenterazine derivative and an apoprotein comprising a peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11;

Step (2): contact said holoprotein with a solution suspected of having calcium ions and

Step (3): determine the luminescence of the holoprotein:calcium complex;

wherein the luminescence correlates with the amount of calcium ions in solution.

2. The method according to claim 1 , wherein said apoprotein further comprises at least one of a peptide sequence for purification and a secretory signal peptide sequence.

3. A method for measuring changes in intracellular calcium concentration in response to an external stimuli, said method comprising:

Step (1): providing a host cell that expresses an apoprotein comprising at least one of items (a) and (b):

wherein (a) is a polypeptide comprising the peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, and

wherein (b) is a polypeptide comprising the peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, and which has the ability to bond with at least one peroxide selected from the group consisting of a peroxide of coelenterazine and a peroxide of a coelenterazine derivative;

Step (2): providing a peroxide of coelenterazine or a peroxide of a coelenterazine derivative to said host cell to form an intracellular holoprotein comprising the apoprotein and peroxide of coelenterazine and a peroxide of a coelenterazine derivative;

Step (3): providing an external stimuli to said host cell;

Step (4): determine the luminescence of the holoprotein:calcium complex;

wherein the luminescence correlates with the ability of the external stimuli to increase or decrease intracellular calcium concentration.

4. A method for determining the transcriptional activation of a promoter by a candidate compound, said method comprising:

Step (1): providing a vector comprising a target promoter operatively linked to a polynucleotide encoding a reporter apoprotein comprising at least one of items (a) and (b),wherein:

(a) is a polypeptide comprising the peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, and

wherein (b) is a polypeptide comprising the peptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, and which has the ability to bond with at least one peroxide selected from the group consisting of a peroxide of coelenterazine and a peroxide of a coelenterazine derivative;

Step (2): transforming a host cell with said vector;

Step (3): culturing said host cell under conditions which allow the expression of the apoprotein;

Step (4): contacting the host cell with a candidate compound;

Step (5): isolating or extracting the apoprotein from the host cell or culture broth;

Step (6): contacting the isolated or extracted apoprotein with at least one peroxide selected from the group consisting of a peroxide of coelenterazine and a peroxide of a coelenterazine derivative to form a holoprotein;

Step (7): contact the holoprotein with calcium;

Step (8): determine the luminescence of the holoprotein:calcium complex; wherein the luminescence correlates with the ability of the candidate compound to activate said promoter.

5. The method according to claim 4 , wherein said polynucleotide is at least one of items (a) and (b):

wherein (a) is a polynucleotide comprising the nucleotide sequence selected from the group consisting of a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and

wherein (b) is a polynucleotide comprising the nucleotide sequence selected from the group consisting of a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and encoding a protein having the ability to bond with at least one peroxide selected from the group consisting of a peroxide of coelenterazine and a peroxide of a coelenterazine derivative so as to form a holoprotein that emits light under the action of a calcium ion.