# US 11,268,106 B2 · Method for synthetic genes

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- Number: US 11,268,106 B2
- Document ID: US11268106B2
- Status: Granted
- Kind: B2
- Application number: 16/216,228
- Filed: 2018-12-11
- Published: 2019-04-04
- Granted: 2022-03-08
- Earliest claimed: 2014-09-11
- Assignees: Jnc Corporation
- Applicants: Jnc Corporation
- Inventors: Satoshi Inouye
- Web page: https://patentgrep.com/patent/US11268106B2

## CPC

- C12N 15/85: for animal cells
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12Y 113/12: with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- 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
- C12Y 113/12013: Oplophorus-luciferin 2-monooxygenase (1.13.12.13)
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present invention provides a method of designing an optimized gene which comprises altering a nucleotide sequence of a target protein gene, so that only preferential codons with high frequency of use in human cells are selected and a GC content of not less than 60% is achieved. A gene design method which involves the feature “only preferential codons with high frequency of use are selected and a GC content of not less than 60% is achieved” can be established as a general rule for preparing proteins with high expression level, in order to obtain chemically synthesized genes for proteins capable of high-level expression in eukaryotes.

## Claims

### Claim 1

An isolated polynucleotide comprising the sequence of SEQ ID NO: 16.

### Claim 2 (depends on 1)

A recombinant expression vector comprising the polynucleotide of claim 1, wherein the sequence of SEQ ID NO: 16 is under the control of a promoter capable of functioning in a mammalian cell.

### Claim 3 (depends on 1)

An isolated recombinant mammalian cell comprising the polynucleotide of claim 1.

### Claim 4 (depends on 3)

The isolated recombinant mammalian cell of claim 3, wherein the mammalian cell is a human cell.

### Claim 5 (depends on 1)

The polynucleotide of claim 1, further comprising a coding sequence of another protein, wherein the polynucleotide encodes a Japanese firefly Luciola cruciata luciferase fusion protein.

### Claim 6 (depends on 2)

A method of producing a Japanese firefly Luciola cruciata luciferase protein, comprising:
introducing the recombinant expression vector of claim 2 into a mammalian cell to produce a recombinant mammalian cell, and
culturing the recombinant mammalian cell to produce the Japanese firefly Luciola cruciata luciferase protein encoded by the sequence of SEQ ID NO: 16.

### Claim 7 (depends on 6)

The method of claim 6, wherein the mammalian cell is a human cell.

### Claim 8 (depends on 5)

A method of producing a Japanese firefly Luciola cruciata luciferase fusion protein, comprising:
preparing a recombinant expression vector comprising the polynucleotide of claim 5 under the control of a promoter capable of functioning in a mammalian cell,
introducing the recombinant expression vector into a mammalian cell to produce a recombinant mammalian cell, and
culturing the recombinant mammalian cell to produce the Japanese firefly Luciola cruciata luciferase fusion protein.

### Claim 9 (depends on 2)

A method of determining luminescence activity, comprising:
introducing the recombinant expression vector of claim 2 into a mammalian cell to produce a recombinant mammalian cell,
culturing the recombinant mammalian cell to produce the Japanese firefly Luciola cruciata luciferase protein encoded by the sequence of SEQ ID NO: 16,
contacting the Japanese firefly Luciola cruciata luciferase with luciferin or a luciferin analogue, and
determining a quantity of light generated by the contacting.

### Claim 10 (depends on 9)

The method of claim 9, wherein the mammalian cell is a human cell.

### Claim 11

A kit comprising:
a) at least one of (i) an isolated polynucleotide comprising the sequence of SEQ ID NO: 16, (ii) a recombinant expression vector comprising a polynucleotide comprising the sequence of SEQ ID NO: 16 under the control of a promoter capable of functioning in a mammalian cell, and (iii) an isolated recombinant mammalian cell comprising a polynucleotide comprising the sequence of SEQ ID NO: 16, and
b) luciferin and/or a luciferin analogue.