# US 8,569,003 B2 · Fusion protein having luminescence activity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,569,003 B2 - Document ID: US8569003B2 - Status: Granted - Kind: B2 - Application number: 13/908,686 - Filed: 2013-06-03 - Published: 2013-09-26 - Granted: 2013-10-29 - Earliest claimed: 2009-12-24 - Assignees: Jnc Corporation - Applicants: Satoshi Inouye; Yuiko Sahara; Junichi Sato - Inventors: Satoshi Inouye; Yuiko Sahara; Junichi Sato - Web page: https://patentgrep.com/patent/US8569003B2 ## CPC - G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)} - C07K 14/43509: {from crustaceans} - C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)} - C12Q 1/66: involving luciferase - C07K 2319/00: Fusion polypeptide - C07K 2319/20: containing a tag with affinity for a non-protein ligand - C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 1) The method according to claim 1, wherein the fusion protein consisting of an amino acid sequence of SEQ ID NO: 4, 6 or 8.