# US 10,308,973 B1 · Real-time imaging method of NAPDH-dependent metabolic activity in cell and use thereof

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- Number: US 10,308,973 B1
- Document ID: US10308973B1
- Status: Granted
- Kind: B1
- Application number: 16/131,951
- Filed: 2018-09-14
- Granted: 2019-06-04
- Earliest claimed: 2018-07-20
- Assignees: Industry Foundation of Chonnam National University
- Applicants: Industry Foundation of Chonnam National University
- Inventors: Geun-Joong Kim; Eung Sam Kim; Jeong Sun Kim; Sung Hwan You; Dae Eun Cheong; Eun Seo Jo
- Web page: https://patentgrep.com/patent/US10308973B1

## CPC

- C12Q 1/32: involving dehydrogenase
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES

## Abstract

Provided is a real-time imaging method of NAPDH-dependent metabolic activity in a cell and a use thereof, and more particularly, an imaging method capable of measuring concentration gradient and change in an intracellular coenzyme using a mutant of metagenome-derived blue fluorescent protein (mBFP) in real-time and an application method thereof. In particular, the method of imaging and quantifying the coenzyme contained in various biological samples may be performed by improving fluorescence in a short time even under anaerobic conditions, without adding a substrate required for coenzyme measurement or conversion without destroying cells, and without consuming the time required for formation of a special structure (fluorophore) for generating fluorescence unlike the existing quantitative systems. In particular, provided is a stable quantitative/imaging technique that is directly applicable to biological samples having various complex compositions without signal interference caused by impurities by removing an inherent enzymatic activity of mBFP.

## Claims

### Claim 1

A mutant of a blue fluorescent protein metagenome-derived blue fluorescent protein mBFP consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 2 (depends on 1)

A nucleic acid sequence encoding the amino acid sequence of the mutant of claim 1.

### Claim 3 (depends on 2)

A recombinant expression vector comprising the nucleic acid sequence of claim 2.

### Claim 4 (depends on 3)

A transformant, excluding human, transformed with the recombinant expression vector of claim 3.

### Claim 5 (depends on 4)

The transformant of claim 4, wherein the transformant is a microorganism, an animal cell, or a plant cell.

### Claim 6 (depends on 5)

The transformant of claim 5, wherein the transformant is Escherichia coli, Pichia pastoris, HeLa cell, or callus cell.

### Claim 7

A method for quantifying NADP(H)- comprising:
adding a mutant of a blue fluorescent protein mBFP consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to a NADP(H)-dependent oxidase/reductase, and measuring a fluorescence change.

### Claim 8 (depends on 7)

The method of claim 7, wherein the NADP(H) is quantified in real time.

### Claim 9 (depends on 7)

The method of claim 7, wherein the amount of NADP(H) is correlated from quantifying a fluorescence change in NADP(H) oxidase/reductase to which the mutant of blue fluorescent protein mBFP has been added.

### Claim 10 (depends on 7)

The method of claim 7, wherein the NADP(H) is quantified in vivo.

### Claim 11

A kit for real-time quantification of NADP(H) comprising:
a mutant of a blue fluorescent protein mBFP consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 12 (depends on 11)

The kit of claim 11, wherein the real time means that analysis is performed until 1 to 15 seconds elapse.