# US 10,988,741 B2 · Mutated genes for the catalytic protein of Oplophorus luciferase and use thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,988,741 B2 - Document ID: US10988741B2 - Status: Granted - Kind: B2 - Application number: 16/403,654 - Filed: 2019-05-06 - Published: 2019-08-22 - Granted: 2021-04-27 - Earliest claimed: 2014-04-16 - Assignees: Jnc Corporation - Applicants: Jnc Corporation - Inventors: Satoshi Inouye; Yuiko Miura; Junichi Sato - Web page: https://patentgrep.com/patent/US10988741B2 ## CPC - C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)} - C12Q 1/66: involving luciferase - C12Y 113/12: with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12) - C12Y 113/12013: Oplophorus-luciferin 2-monooxygenase (1.13.12.13) - 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 - C12Y: ENZYMES ## Abstract A novel luciferase that is distinct from conventional luciferase has been desired. A luciferase mutant comprising an amino acid sequence in which glutamic acid at position 4, arginine at position 11, leucine at position 18 and valine at position 27 are substituted with other amino acids, in the amino acid sequence of SEQ ID NO: 2. ## Claims ### Claim 1 A method for producing a recombinant luciferase mutant, which comprises the steps of culturing a transformant transformed with a recombinant vector comprising a polynucleotide comprising a nucleotide sequence encoding the recombinant luciferase mutant and separating/purifying the recombinant luciferase mutant, wherein the recombinant luciferase mutant is selected from the group consisting of: (a) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the amino acid positions consisting of: glutamic acid at position 4; arginine at position 11; leucine at position 18; and valine at position 27; and, (b) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of: glutamic acid at position 4; arginine at position 11; leucine at position 18; valine at position 27; and one or more amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 2, wherein the recombinant luciferase mutant has luciferase activity. ### Claim 2 (depends on 1) The method of claim 1, wherein the recombinant luciferase mutant defined in (b) is a recombinant luciferase mutant defined in (c) below: (c) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of: glutamic acid at position 4; arginine at position 11; leucine at position 18; valine at position 27; and 1 to 16 amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 2, and wherein the recombinant luciferase mutant has luciferase activity. ### Claim 3 (depends on 1) The method of claim 1, wherein glutamic acid at the amino acid position 4 is substituted with alanine, arginine at the amino acid position 11 is substituted with glutamine, leucine at the amino acid position 18 is substituted with glutamine, and valine at the amino acid position 27 is substituted with leucine. ### Claim 4 (depends on 1) The method of claim 1, wherein the recombinant luciferase mutant defined in (a) or (b) is a recombinant luciferase mutant defined in (d) or (e), respectively, below: (d) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4; or, (e) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid(s) is/are substituted with other amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 4, and wherein the recombinant luciferase mutant has luciferase activity. ### Claim 5 (depends on 4) The method of claim 4, wherein the luciferase mutant defined in (e) is a recombinant luciferase mutant defined in (f) below: (f) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4 in which 1 to 16 amino acid(s) is/are substituted with other amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 4, and wherein the recombinant luciferase mutant has luciferase activity. ### Claim 6 A method for assaying the activity of a target promoter, which comprises providing a polynucleotide encoding a luciferase mutant as a report gene and fusing the polynucleotide to the target promoter to construct a vector; transforming the vector into a host cell; contacting the host cell with a luciferin; and detecting luminescence, thereby assaying the activity of the target promoter, wherein the luciferase mutant is selected from the group consisting of: (a) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the amino acid positions consisting of: glutamic acid at position 4; arginine at position 11; leucine at position 18; and valine at position 27; and, (b) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of: glutamic acid at position 4; arginine at position 11; leucine at position 18; valine at position 27; and one or more amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 2, wherein the recombinant luciferase mutant has luciferase activity. ### Claim 7 (depends on 6) The method of claim 6, wherein the luciferin is a coelenterazine analogue. ### Claim 8 (depends on 7) The method of claim 7, wherein the coelenterazine analogue is coelenterazine, h-coelenterazine or f-coelenterazine. ### Claim 9 (depends on 6) The method of claim 6, wherein the recombinant luciferase mutant defined in (b) is a recombinant luciferase mutant defined in (c) below: (c) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 2 substituted at the positions consisting of: glutamic acid at position 4; arginine at position 11; leucine at position 18; valine at position 27; and 1 to 16 amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 2, and wherein the recombinant luciferase mutant has luciferase activity. ### Claim 10 (depends on 6) The method of claim 6, wherein glutamic acid at the amino acid position 4 is substituted with alanine, arginine at the amino acid position 11 is substituted with glutamine, leucine at the amino acid position 18 is substituted with glutamine, and valine at the amino acid position 27 is substituted with leucine. ### Claim 11 (depends on 6) The method of claim 6, wherein the recombinant luciferase mutant defined in (a) or (b) is a recombinant luciferase mutant defined in (d) or (e), respectively, below: (d) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4; or, (e) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4 in which one or more amino acid(s) is/are substituted with other amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 4, and wherein the recombinant luciferase mutant has luciferase activity. ### Claim 12 (depends on 11) The method of claim 11, wherein the luciferase mutant defined in (e) is a recombinant luciferase mutant defined in (f) below: (f) a recombinant luciferase mutant comprising the amino acid sequence of SEQ ID NO: 4 in which 1 to 16 amino acid(s) is/are substituted with other amino acid(s) at position(s) other than positions 4, 11, 18, 27, 33, 43, 44, 54, 68, 72, 75, 90, 115, 124, 138 and 166 of the amino acid sequence of SEQ ID NO: 4, and wherein the recombinant luciferase mutant has luciferase activity.