# US 11,999,978 B2 · Alkaline phosphatase composition, method of producing dephosphorylated nucleic acid and method of producing labeled nucleic acid

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- Number: US 11,999,978 B2
- Document ID: US11999978B2
- Status: Granted
- Kind: B2
- Application number: 17/279,686
- Filed: 2019-09-25
- Published: 2021-12-23
- Granted: 2024-06-04
- Earliest claimed: 2018-09-25
- Assignees: Toray Industries, Inc.
- Applicants: Toray Industries, Inc.
- Inventors: Masateru Ito; Yoji Ueda; Yuki Takii; Mai Yagi
- Web page: https://patentgrep.com/patent/US11999978B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Y 301/03001: Alkaline phosphatase (3.1.3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

A composition contains an alkaline phosphatase and first to sixth peptide fragments, wherein content ratios of the first to sixth peptide fragments to the alkaline phosphatase satisfy formulas (1) to (6), respectively: (X1/Y)×100≤0.6000 (1); (X2/Y)×100≤0.1800 (2); (X3/Y)×100≤0.2000 (3); (X4/Y)×100≤0.8000 (4); (X5/Y)×100≤1.6000 (5); and (X6/Y)×100≤0.3500 (6), wherein X1 to X6 represent peak area values of the first to sixth peptide fragments calculated by an automatic integration method from an extracted ion chromatogram obtained by an LC-MS/MS analysis of the composition, respectively, and Y represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by an LC-UV analysis of the composition.

## Claims

### Claim 1

A composition comprising:
an alkaline phosphatase;
a first peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 1;
a second peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2;
a third peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 3;
a fourth peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 4;
a fifth peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 5; and
a sixth peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 6,
wherein content ratios of the first to sixth peptide fragments to the alkaline phosphatase satisfy formulas (1) to (6), respectively:
(X1/Y)×100≤0.6000 (1);
(X2/Y)×100≤0.1800 (2);
(X3/Y)×100≤0.2000 (3);
(X4/Y)×100≤0.8000 (4);
(X5/Y)×100≤1.6000 (5); and
(X6/Y)×100≤0.3500 (6),
wherein X1 to X6 represent peak area values of the first to sixth peptide fragments calculated by an automatic integration method from an extracted ion chromatogram obtained by an LC-MS/MS analysis of the composition, respectively, and Y represents a peak area value of the alkaline phosphatase calculated by an automatic integration method from a chromatogram obtained by an LC-UV analysis of the composition.

### Claim 2 (depends on 1)

The composition according to claim 1, wherein:
the composition further comprises a seventh peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 7; and
a content ratio of the seventh peptide fragment to the alkaline phosphatase satisfies formula (7):
(X7/Y)×100≤1.0000 (7),
wherein X7 represents a peak area value of the seventh peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by an LC-MS/MS analysis of the composition, and Y is the same as defined above.

### Claim 3 (depends on 1)

The composition according to claim 1, wherein:
the composition further comprises an eighth peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 8; and
a content ratio of the eighth peptide fragment to the alkaline phosphatase satisfies formula (8):
(X8/Y)×100≤1.0000 (8),
wherein X8 represents a peak area value of the eighth peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by an LC-MS/MS analysis of the composition, and Y is the same as defined above.

### Claim 4 (depends on 1)

The composition according to claim 1, wherein:
the composition further comprises a ninth peptide fragment consisting of the amino acid sequence set forth in SEQ ID NO: 9; and
a content ratio of the ninth peptide fragment to the alkaline phosphatase satisfies formula (9):
(X9/Y)×100≤2.3000 (9),
wherein X9 represents a peak area value of the ninth peptide fragment calculated by an automatic integration method from an extracted ion chromatogram obtained by an LC-MS/MS analysis of the composition, and Y is the same as defined above.

### Claim 5 (depends on 1)

The composition according to claim 1, wherein the composition has an alkaline phosphatase specific activity of 2,000 U/mg or more.

### Claim 6 (depends on 1)

The composition according to claim 1, wherein the alkaline phosphatase is selected from the following (a) and (b):
(a) an alkaline phosphatase comprising a protein molecule consisting of the amino acid sequence set forth in SEQ ID NO: 10; and
(b) an alkaline phosphatase comprising a protein molecule consisting of an amino acid sequence that has 70% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 10 and comprises positions 78 to 90, positions 177 to 187, positions 469 to 477, positions 516 to 528 and positions 534 to 551 of the amino acid sequence set forth in SEQ ID NO: 10.

### Claim 7 (depends on 6)

The composition according to claim 6, wherein the amino acid sequence of the protein molecule of the alkaline phosphatase (b) further comprises one or two or more selected from positions 91 to 109, positions 93 to 105 and positions 529 to 531 of the amino acid sequence set forth in SEQ ID NO: 10.

### Claim 8 (depends on 6)

The composition according to claim 6, wherein the amino acid sequence of the protein molecule of the alkaline phosphatase (b) further comprises positions 91 to 109 and positions 529 to 531 of the amino acid sequence set forth in SEQ ID NO: 10.

### Claim 9 (depends on 1)

The composition according to claim 1, wherein the composition further comprises a nucleic acid, and is a composition used for dephosphorylating the nucleic acid.

### Claim 10 (depends on 1)

The composition according to claim 1, wherein the composition further comprises a dephosphorylated nucleic acid, and is a composition used for preparing a labeled nucleic acid comprising the dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid.

### Claim 11 (depends on 1)

The composition according to claim 1, wherein the composition further comprises a labeled nucleic acid comprising a dephosphorylated nucleic acid and a labeling substance bound to the dephosphorylated nucleic acid, and is a nucleic acid sample to be subjected to a nucleic acid detection method.

### Claim 12 (depends on 11)

The composition according to claim 11, wherein the nucleic acid detection method is a nucleic acid detection method using a nucleic acid microarray.

### Claim 13 (depends on 1)

A method of producing a dephosphorylated nucleic acid, the method comprising:
providing the composition according to claim 1;
providing a nucleic acid; and
treating the nucleic acid with the composition to dephosphorylate the nucleic acid.

### Claim 14 (depends on 1)

A method of producing a labeled nucleic acid, the method comprising:
providing the composition according to claim 1;
providing a nucleic acid;
providing a labeling substance;
treating the nucleic acid with the composition to dephosphorylate the nucleic acid; and
binding the labeling substance to the dephosphorylated nucleic acid.