# US 10,371,708 B2 · Metal ion sensors and methods of detecting metal ions

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- Number: US 10,371,708 B2
- Document ID: US10371708B2
- Status: Granted
- Kind: B2
- Application number: 14/908,684
- Filed: 2014-08-06
- Published: 2016-08-25
- Granted: 2019-08-06
- Earliest claimed: 2013-08-06
- Assignees: Georgia State University Research Foundation, Inc.
- Applicants: Georgia State University Research Foundation, Inc.
- Inventors: Jenny Jie Yang; Shen Tang; You Zhuo
- Web page: https://patentgrep.com/patent/US10371708B2

## CPC

- G01N 33/84: involving inorganic compounds or pH
- C07K 14/43595: {from coelenteratae, e.g. medusae}
- C07K 14/4728: {Calcium binding proteins, e.g. calmodulin}
- 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 33/582: {with fluorescent label}
- C07K 2319/04: containing an ER retention signal such as a C-terminal HDEL motif
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- G01N 2021/6441: {with two or more labels}
- 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)
- 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

A method for constructing an metal ion binding motif by identifying an metal ion binding peptide that binds an metal ion with specificity, ascertaining at least a portion of a nucleic acid sequence encoding the metal ion binding peptide, tailoring the nucleic acid sequence encoding the metal ion binding peptide into an metal ion binding site, identifying a host protein and a relevant portion of the nucleic acid sequence of the host protein, operatively linking the tailored nucleic acid sequence encoding the metal ion binding peptide and the host protein nucleic acid sequence into an metal ion binding motif sequence, and expressing metal ion binding motif sequence, in which the nucleic acid sequence encoding the metal ion binding peptide is tailored so as to achieve the metal ion binding motif with a desired specificity for the metal ion. Also, the proteins encoded by the metal ion binding motif sequence as constructed by the method.

## Claims

### Claim 1

A polypeptide metal ion sensor comprising an engineered green fluorescent polypeptide (GFP) having a heterologous metal ion binding site, wherein said engineered GFP is a variant amino acid sequence of SEQ ID NO: 37 having the mutations corresponding to L22V and 1218M and, when having a metal ion species bound thereto, has an elevated fluorescence output compared to the polypeptide SEQ ID NO: 37 binding to the same metal ion species at 37° C., wherein the amino acid sequence of said engineered GFP comprises any one of the amino acid sequences selected from the group consisting of sequences having 95% similarity to any one of SEQ ID NOs: 71, 72, 73, 74, 79, 81, and 82, or wherein said engineered GFP has 100% similarity to SEQ ID NO: 80 or said engineered GFP consists of SEQ ID NO: 75.

### Claim 2 (depends on 1)

The polypeptide metal ion sensor of claim 1, wherein said sensor is conjugated to at least one targeting polypeptide motif that specifically recognizes a structural feature of a cell.

### Claim 3 (depends on 2)

The polypeptide metal ion sensor of claim 2, wherein said at least one targeting polypeptide motif specifically recognizes a target component of an endoplasmic reticulum or a sarcoplasmic reticulum of a cell.

### Claim 4 (depends on 3)

The polypeptide metal ion sensor of claim 3, wherein said targeting polypeptide motif has at least 90% sequence identity with an amino acid sequence selected from the group consisting of the sequences SEQ ID NOs: 64, 76-78, and the sequence KDEL (SEQ ID NO. 83).

### Claim 5 (depends on 1)

The polypeptide metal ion sensor of claim 1, wherein said metal ion binding site specifically binds to a metal ion selected from the group consisting of: calcium, lead, gadolinium, lanthanum, terbium, antimony, strontium, magnesium, mercury, and cadmium.

### Claim 6

A method of detecting metal ions in a biological sample, comprising: (i) providing a polypeptide metal ion sensor comprising an engineered green fluorescent polypeptide (GFP) having a heterologous metal ion binding site, wherein said engineered GFP is a variant of amino acid sequence SEQ ID NO: 37 and having the amino acid substitutions corresponding to L22V and I218M and, when having a metal ion species bound thereto, has an elevated fluorescence output compared to the polypeptide SEQ ID NO: 37 binding to the same metal ion species at 37° C., wherein the amino acid sequence of said engineered GFP comprises any one of the amino acid sequences selected from the group consisting of sequences having 95% similarity to any one of SEQ ID NOs: 71, 72, 73, 74, 79, 81, and 82, or wherein said engineered GFP has 100% similarity to SEQ ID NO: 80 or said engineered GFP consists of SEQ ID NO: 75; (ii) delivering the polypeptide metal ion sensor or an expression vector having an nucleic acid sequence encoding said metal sensor to a biological sample; (iii) detecting a first fluorescent signal emitted by said sensor; (iii) generating a physiological or cellular change in the biological sample; (iv) detecting a second fluorescent signal emitted by said sensor after step (iii); and (v) comparing the first and second fluorescent signals, wherein a ratiometric change in at least one of a wavelength, an intensity, and lifetime between the first and second fluorescent signals indicates a change in the rate of release or intracellular concentration of a metal ion in the sample.

### Claim 7 (depends on 6)

The method of claim 6, wherein the ratiometric change in the signal intensity provides an quantitative measurement of the metal ion in the sample.

### Claim 8 (depends on 6)

The method of claim 6, wherein the biological sample is a cell or tissue of an animal or human subject, or a cell or tissue isolated from an animal or human subject.

### Claim 9 (depends on 6)

The method of claim 6, wherein the fluorescence signal generated when a metal ion is bound to said sensor is used to generate an image.