# US 8,772,454 B2 · Isolation and characterization of novel green fluorescent proteins from copepods

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- Number: US 8,772,454 B2
- Document ID: US8772454B2
- Status: Granted
- Kind: B2
- Application number: 13/256,915
- Filed: 2010-03-10
- Published: 2012-03-22
- Granted: 2014-07-08
- Earliest claimed: 2009-03-20
- Assignees: Board of Regents, The University of Texas System
- Applicants: Mikhail V. Matz; Marguerite E. Hunt
- Inventors: Mikhail V. Matz; Marguerite E. Hunt
- Web page: https://patentgrep.com/patent/US8772454B2

## CPC

- C07K 14/43595: {from coelenteratae, e.g. medusae}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The isolation and characterization of two protein isoforms collected green fluorescent copepods is described herein. The new Pontella mimocerami GFP-like isoforms pmimGFP1 and pmimGFP2 of the present invention are quick to mature and rapidly produce a fluorescent signal. The two isoforms are very similar in molar extinction coefficients (ME) with 105,000 M−1 cm−1 for pmimGFP1 and 103,000 M−1cm−1 for pmimGFP2, respectively. The relative brightness of these two new copepod GFP-like proteins is the highest measured for any isolated GFP-like protein.

## Claims

### Claim 1

An isolated protein molecule comprising an amino acid sequence, wherein the amino acid sequence is at least 96% identical to the amino acid sequence of SEQ ID NO: 1 or wherein the amino acid sequence is encoded by a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of SEQ ID NO: 2, and wherein the isolated protein molecule has green fluorescent protein activity.

### Claim 2 (depends on 1)

The protein molecule of claim 1, wherein the protein molecule has a molar extinction coefficient of at least 100,000 M−1cm−1.

### Claim 3 (depends on 1)

The protein molecule of claim 1, wherein the protein molecule has a quantum yield of at least 0.90.

### Claim 4 (depends on 1)

The protein molecule of claim 1, wherein the amino acid sequence is at least 97, 98 or 99% identical to the amino acid sequence of SEQ ID NO: 1 or wherein the amino acid sequence is encoded by a nucleic acid sequence that is at least 97, 98 or 99% identical to the nucleic acid sequence of SEQ ID NO: 2.

### Claim 5 (depends on 1)

The protein molecule of claim 1, wherein the protein molecule has an absorbance maximum between 480-490 nm and an emission maximum between 500-511 nm.

### Claim 6 (depends on 1)

The protein molecule of claim 1, wherein the protein molecule comprises the amino acid sequence of SEQ ID NO: 1 and is isolated from Pontella mimocerami.

### Claim 7

A purified green fluorescent protein produced by
ligating one or more amplified nucleic acids encoding a green fluorescent protein into a vector, wherein said one or more amplified nucleic acids have a nucleotide sequence with at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 2, and wherein the encoded green fluorescent protein has green fluorescent protein activity;
transfecting a host cell with the vector;
expressing the green fluorescent protein in the host cell; and
purifying the green fluorescent protein from the host cell.

### Claim 8

An isolated green fluorescent protein isolated by
transfecting one or more cells with a nucleic acid encoding a green fluorescent protein, wherein said nucleic acid has a nucleotide sequence with at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 2 and wherein the green fluorescent protein has green fluorescent protein activity;
plating the one or more cells onto a culture plate;
incubating the culture plate at 37° C. for a period not less than 8 hours, to express said green fluorescent protein in the one or more cells and form one or more green fluorescent colonies;
selecting and picking at least one of the one or more green fluorescent colonies from the culture plate to form selected green fluorescent colonies;
resuspending the selected green fluorescent colonies in water or a buffer to form resuspended green fluorescent colonies;
plating the resuspended green fluorescent colonies onto a second culture plate;
incubating the second culture plate at room temperature for at least 48 hours to form green fluorescent colonies;
resuspending the green fluorescent colonies in the buffer;
sonicating the green fluorescent colonies in the buffer on ice to form a sonicated buffer solution;
centrifuging the sonicated buffer solution;
separating the sonicated buffer solution to yield a lysate and a cellular debris; and
isolating said green fluorescent protein from the lysate, the cellular debris or both using metal-affinity chromatography, chromatography, affinity extraction, or other protein purification techniques.

### Claim 9

A kit comprising one or more containers, wherein at least one of the one or more containers comprises an isolated protein molecule comprising an amino acid sequence, wherein the amino acid sequence is at least 96% identical to the amino acid sequence of SEQ ID NO: 1, and wherein the isolated protein molecule has green fluorescent protein activity.