Recombinant photoproteins and their conjugates
This invention is to provide a photoprotein which binds with a ligand specific for a substance to be detected at a binding ratio of 1:1 such that the luminescence activity is not reduced by binding with the ligand, a conjugate comprising the luminescent photoprotein and ligand, and a substance detection method which employs the conjugate as a marker. A calcium-binding photoprotein is produced having cysteine residue introduced within the 4th amino acid residue from the amino-terminus. A conjugate is formed by binding a ligand specific for a substance to be detected to the calcium-binding photoprotein, in a binding ratio of 1:1, via the introduced cysteine residue. The conjugate may be utilized as a marker for a substance to be detected.
1. A recombinant calcium-binding photoprotein aequorin, comprising a mutant apoaequorin having the amino acid sequence listed as SEQ. ID. No.2.
2. The recombinant calcium-binding photoprotein aequorin according to claim 1 , comprising as luminescent substrate coelenterazine or its analog which exhibits the luminescence activity.
3. A conjugate, which comprises a recombinant calcium-binding photoprotein aequorin and a ligand specific for a substance to be detected, wherein said recombinant calcium-binding photoprotein aequorin comprises a mutant apoaequorin having the amino acid sequence listed as SEQ. ID. No. 2 and said ligand binds to said recombinant calcium-binding photoprotein aecluorin via the 6th cysteine, in a ratio of the ligand and the recombinant calcium-binding photoprotein aequorin being 1:1.
4. The conjugate according to claim 3 , wherein the ligand specific for a substance to be detected is biotin, avidin, streptavidin, an enzyme, a substrate, an antibody, an antigen, nucleic acid, a polysaccharide, a receptor or a compound capable of binding to any of these.
5. A kit for measuring a substance specific for a ligand, comprising a conjugate according to claim 4 .
6. The conjugate according to claim 3 , comprising as luminescent substrate coelenterazine or its analog which exhibits the luminescence activity.
7. A kit for measuring a substance specific for a ligand, comprising a conjugate according to claim 6 .
8. The conjugate according to claim 3 , wherein the ligand specific for a substance to be detected is biotin.
9. A kit for measuring a substance specific for a ligand, comprising a conjugate according to claim 8 .
10. A method for measuring a substance specific for a ligand, characterized by using a conjugate according to claim 3 .
11. A kit for measuring a substance specific for a ligand, comprising a conjugate according to claim 3 .
12. A kit for measuring a substance specific for a ligand, comprising a conjugate according to claim 3 .
13. A method for producing a conjugate, which comprises producing by genetic engineering a mutant apoaequorin having the amino acid sequence listed as SEQ. ID. No.2, treating the apoaequorin with coelenterazine in the presence of oxygen to yield a recombinant calcium-binding photoprotein, and binding the photoprotein via the 6th cysteine to a ligand specific for a substance to be detected, in a ratio of the ligand and the recombinant calcium-binding photoprotein aequorin being 1:1.
14. The method for producing a conjugate according to claim 13 , wherein the ligand specific for a substance to be detected is biotin.