Recombinant photoproteins and their conjugates

Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues

Reexamination Certificate

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C435S007500, C435S069100, C435S320100, C435S325000

Reexamination Certificate

active

10400630

ABSTRACT:
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.

REFERENCES:
Lewis and Daunert. Anal Chem. 2001; 73: 3227-3233.
Paolo F. Zatta, et al. “A Solid-Phase Assay For β-1, 4-Galactosyltransferase Activity in Human Serum Using Recombinant Aequorin” Analytical Biochemistry 194, 1991, pp. 185-191.
J. C. Lewis, et al. Site-Specifically Labeled Photoprotein-Thyroxine Conjugates Using Aequorin Mutants Containing Unique Cysteine Residues: Applications For Binding Assays (Part II) Bioconjugate Chem. 11, 2000, pp. 140-145.
Satoshi Inouye, et al. “Cloning and Sequence Analysis of cDNA for the Luminescent Protein Aequorin” Proc. Natl. Acad. Sci USA, vol. 82, May 1985, pp. 3154-3158.
James F. Head, et al. “The Crystal Structure of the Photoprotein Aequorin at 2.3 Å Resolution” Nature, vol. 405, May 18, 2000, pp. 372-376.
Osamu Shimomura, et al. “Mechanism of the Luminescent Intramolecular Reaction of Aequorin,” Biochemistry, vol. 13, No. 16, 1974, pp. 3278-3286.
Kouichi Kurose, et al. “Bioluminescence of the Ca2+-Binding Photoprotein Aequorin After Cystein Modification” Proc. Natl. Acad. Sci. USA, vol. 86, Jan. 1989, pp. 80-84.
J. C. Lewis, et al. “Bioluminescence and Secondary Structure Properties of Aequorin Mutants Produced for Site-Specific Cunjugation and Immobilization” Bioconjugate Chem. 11, 2000 pp. 65-70.
Midori Nomura, et al. “A C-Terminal Proline is Required for Bioluminescence of the Ca2+-Binding Photoprotein, Aequorin” Federation of European Biochemical Societies, vol. 295, No. 1,2,3, Dec. 1999, pp. 63-66.
Satoshi Inouye, et al. “Overexpression and Purification of the Recombinant Ca2+-Binding Protein, Apoaequorin” J. Biochem. 105, 1989, pp. 473-477.
Satoshi Inouye, et al. “High-Level Expression and Purification of Apoaequorin” Protein and Expression and Purification 2, 1991, pp. 122-126.
Osamu Shimomura, et al. “The Relative Rate of Aequorin Regeneration From Apoaequorin and Coelenterazine Analogues” Biochem. J. 296, 1993, pp. 549-551.

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