# US 12,215,375 B2 · Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use

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- Number: US 12,215,375 B2
- Document ID: US12215375B2
- Status: Granted
- Kind: B2
- Application number: 17/332,459
- Filed: 2021-05-27
- Published: 2022-11-03
- Granted: 2025-02-04
- Earliest claimed: 2010-11-02
- Assignees: Promega Corporation
- Applicants: Promega Corporation
- Inventors: Brock Binkowski; Lance P. Encell; Mary Hall; Matthew B. Robers; Michael R. Slater; Keith V. Wood; Monika G. Wood
- Web page: https://patentgrep.com/patent/US12215375B2

## CPC

- C12Q 1/66: involving luciferase
- C07D 487/04: Ortho-condensed systems
- C12N 9/0004: {Oxidoreductases (1.)}
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12Y 113/12005: Renilla-luciferin 2-monooxygenase (1.13.12.5), i.e. renilla-luciferase
- C12Y 113/12007: Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
- A01K 2217/072: maintaining or altering function, i.e. knock in
- 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
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

An isolated polynucleotide encoding a modified luciferase polypeptide and substrates. The OgLuc variant polypeptide has at least 60% amino acid sequence identity to SEQ ID NO: 1 and at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID NO: 1. The OgLuc variant polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the corresponding polypeptide of the wild-type Oplophorus luciferase.

## Claims

### Claim 1

A composition comprising a circularly permuted polypeptide having an N-terminal segment linked to a C-terminal segment, wherein the N-terminal segment of the circularly permuted polypeptide has at least 96% sequence identity to a portion of SEQ ID NO: 89 from a circular permutation site to the C-terminus of SEQ ID NO: 89, wherein the C-terminal segment of the circularly permuted polypeptide has at least 96% sequence identity to a portion of SEQ ID NO: 89 from the N-terminus to the circular permutation site of SEQ ID NO: 89, wherein the circular permutation site is at a position corresponding to one of positions 27, 122, 147, and 157 of SEQ ID NO: 89, and wherein the circularly permuted polypeptide exhibits luciferase activity in the presence of a coelenterazine substrate.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the N-terminal and C-terminal segments of the circularly permuted polypeptide are fused together by a peptide linker.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the peptide linker comprises a protease cleavage site.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the N-terminal and C-terminal segments of the circularly permuted polypeptide together have 100% sequence identity to the amino acid sequence of SEQ ID NO: 89.

### Claim 5 (depends on 1)

The composition of claim 1, further comprising a coelenterazine substrate.