Fusion protein having luminescence activity
The fusion protein comprising (1) a first region comprising the amino acid sequence of SEQ ID NO: 18 and (2) a second region comprising an amino acid sequence for a polypeptide containing at least one cysteine residue for binding to other useful compound via the thiol group can be modified by chemical modification, and thus has a high catalytic ability for a luminescence activity and is highly available for general purposes.
1. A method for determining a substance that specifically binds to a ligand, which comprises binding a test substance via the ligand to a complex comprising a fusion protein comprising:
(1) a first region selected from the group consisting of (a) to (d) below:
(a) a region consisting of the amino acid sequence of SEQ ID NO: 18;
(b) a region consisting of the amino acid sequence of SEQ ID NO: 18 wherein 1 to 10 amino acids are deleted, substituted, inserted and/or added and having a catalytic ability for a luminescence activity with a luciferin which is a substrate;
(c) a region consisting of an amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 18 and having a catalytic ability for a luminescence activity with a luciferin which is a substrate; and,
(d) a region consisting of an amino acid sequence encoded by a polynucleotide which hybridizes under high stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17 and having a catalytic ability for a luminescence activity with a luciferin which is a substrate, wherein the high stringent conditions are 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50° C.; and,
(2) a second region consisting of an amino acid sequence for a polypeptide having at least one cysteine residue for binding to other useful compound via its thiol group, wherein the second region is selected from the group consisting of (e) to (h) below:
(e) a region consisting of the amino acid sequence of SEQ ID NO: 20;
(f) a region comprising the amino acid sequence of SEQ ID NO: 20 wherein 1 to 3 amino acids are deleted, substituted, inserted and/or added and having at least one cysteine residue for binding to other useful compound via the thiol group;
(g) a region comprising an amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 20 and having at least one cysteine residue for binding to other useful compound via the thiol group; and,
(h) a region comprising an amino acid sequence encoded by a polynucleotide which hybridizes under high stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 19 and having at least one cysteine residue for binding to other useful compound via the thiol group, wherein the high stringent conditions are 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50° C.; and
a ligand bound to the fusion protein via the thiol group of the cysteine residue in the second region and detecting light being emitted from the fusion protein in the presence of a luciferin to determine the substance.
2. The method according to claim 1 , wherein:
(1) the first region is a region consisting of the amino acid sequence of SEQ ID NO: 18, and,
(2) the second region is a region consisting of the amino acid sequence of SEQ ID NO: 20.
3. The method according to claim 1 , wherein the fusion protein further comprises an amino acid sequence for promoting translation and/or an amino acid sequence for purification.
4. The method according to claim 1 , wherein the fusion protein consisting of an amino acid sequence of SEQ ID NO: 4, 6 or 8.