# US 9,518,996 B2 · Fluorescent protein-based calcium integrators

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- Number: US 9,518,996 B2
- Document ID: US9518996B2
- Status: Granted
- Kind: B2
- Application number: 14/452,428
- Filed: 2014-08-05
- Published: 2015-02-05
- Granted: 2016-12-13
- Earliest claimed: 2013-08-05
- Assignees: Howard Hughes Medical Institute
- Applicants: Howard Hughes Medical Institute
- Inventors: Eric R. Schreiter; Loren L. Looger; Benjamin F. Fosque
- Web page: https://patentgrep.com/patent/US9518996B2

## CPC

- G01N 33/84: involving inorganic compounds or pH
- C07K 14/4728: {Calcium binding proteins, e.g. calmodulin}
- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- G01N 33/582: {with fluorescent label}
- C07K 2319/095: containing a nuclear export signal
- C07K 2319/21: containing a His-tag
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The presently-disclosed subject matter includes fluorescent protein-based indicators for detecting ions, small molecule analytes, or combinations thereof. In some embodiments the indicators include a polypeptide, which itself includes a fluorescent polypeptide, a compound-binding polypeptide, and a polypeptide target of the compound-binding polypeptide. In some embodiments the polypeptide includes an EosFP polypeptide, a calmodulin polypeptide, and a M13 polypeptide, or fragments and/or variants thereof. The presently-disclosed subject matter also includes methods for detecting calcium in a sample with embodiments of the present polypeptides. In some embodiments the present indicators experience a permanent shift from green to red fluorescent when exposed to an detecting substance, such as calcium.

## Claims

### Claim 1

An isolated polypeptide, comprising: an EosFP polypeptide, which includes
a C-terminus portion comprising the amino acid sequence of SEQ ID NO. 2, a variant thereof having 95% identity to SEQ ID NO. 2, or a fragment thereof wherein 1-5 amino acids removed relative to SEQ ID NO. 2, and
an N-terminus portion comprising the amino acid sequence of SEQ ID NO. 3, a variant thereof having 95% identity to SEQ ID NO. 3, or a fragment thereof wherein 1-5 amino acids removed relative to SEQ ID NO. 3;
a calmodulin (CaM) polypeptide, comprising the amino acid sequence of SEQ ID NO. 4, a variant thereof having 95% identity to SEQ ID NO. 4, or a fragment thereof wherein 1-5 amino acids removed relative to SEQ ID NO. 4; and
a M13 polypeptide, comprising the amino acid sequence of SEQ ID NO. 5, a variant thereof having mutations at residues of position 2, 6, 9, 10, 11, and/or 13 of SEQ ID NO: 5, or a fragment thereof wherein 1-5 amino acids removed relative to SEQ ID NO. 5.

### Claim 2 (depends on 1)

The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO. 1.

### Claim 3 (depends on 1)

The isolated polypeptide of claim 1, wherein, from an N-terminus to a C-terminus, the isolated polypeptide comprises the CaM polypeptide, the EosFP polypeptide, and the M13 polypeptide.

### Claim 4 (depends on 1)

The isolated polypeptide of claim 1, wherein, from a C-terminus to an N-terminus, the isolated polypeptide comprises the CaM polypeptide, the EosFP polypeptide, and the M13 polypeptide.

### Claim 5 (depends on 1)

The isolated polypeptide of claim 1, further comprising a first polypeptide linker disposed between the CaM polypeptide and the EosFP polypeptide, a second polypeptide linker disposed between the EosFP polypeptide and the M13 polypeptide, an inter-domain linker disposed between the C-terminus portion of the EosFP polypeptide and the N-terminus portion of the EosFP polypeptide, or a combination thereof.

### Claim 6 (depends on 5)

The isolated polypeptide of claim 5, wherein the inter-domain linker comprises about 1 to about 6 amino acids.

### Claim 7 (depends on 5)

The isolated polypeptide of claim 5, wherein the inter-domain linker comprises an amino acid sequence of SEQ ID NO. 13.

### Claim 8 (depends on 5)

The isolated polypeptide of claim 5, wherein the first polypeptide linker includes a 6×His tag.

### Claim 9 (depends on 1)

The isolated polypeptide of claim 1, further comprising a nuclear export signal (NES).

### Claim 10 (depends on 9)

The isolated polypeptide of claim 9, wherein the NES is at a N-terminus of the isolated polypeptide and/or at a C-terminus of the isolated polypeptide.

### Claim 11 (depends on 9)

The isolated polypeptide of claim 9, wherein the NES comprises the sequence of SEQ ID NO: 6.

### Claim 12 (depends on 1)

The isolated polypeptide of claim 1, wherein the M13 polypeptide comprises a variant of the amino acid sequence of SEQ ID NO. 5, wherein the mutations of the variant are selected from the group consisting of S2L, W6Y, W6L, W6V, W6M, W6H, W6F, T9A, T9D, G10D, G10A, H11K, V13H, V13S, V13T, V13A, V13D, and V13L.

### Claim 13 (depends on 1)

The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises a variant of the amino acid sequence of SEQ ID NO. 1, wherein mutations of the variant are selected from the group consisting of Q171V, Q171T, Q171R, M172R, M172L, M173T, A175L, H194W, F227Y, C231R, C231T, C231A, I232V, I232C, L246I, L281V, N374A, and N374S.

### Claim 14 (depends on 1)

The isolated polypeptide of claim 1, wherein the EosFP polypeptide includes a circular permutation on a beta strand of the EosFP polypeptide selected from the beta strand 1, 5, 7, 8, 9, and combinations thereof.

### Claim 15 (depends on 1)

The isolated polypeptide of claim 1, wherein, in the presence of calcium, a fluorescence emitted by the isolated polypeptide shifts from green to red.

### Claim 16 (depends on 1)

The isolated polypeptide of claim 1, wherein the EosFP polypeptide includes a C-terminus portion comprising the amino acid sequence of SEQ ID NO. 2 and a N-terminus portion comprising the amino acid sequence of SEQ ID NO. 3; the calmodulin (CaM) polypeptide comprises the amino acid sequence of SEQ ID NO. 4; and the M13 polypeptide comprises the amino acid sequence of SEQ ID NO. 5.

### Claim 17 (depends on 1)

The isolated polypeptide of claim 1, wherein
the EosFP polypeptide comprises
a C-terminus portion comprising a fragment of the amino acid sequence of SEQ ID NO. 2, wherein the fragment has the amino acid sequence of SEQ ID NO: 2 wherein 1-5 amino acids are removed relative to SEQ ID NO. 2 and
a N-terminus portion comprising a fragment of the amino acid sequence of SEQ ID NO. 3, wherein the fragment has the amino acid sequence of SEQ ID NO: 3 wherein 1-5 amino acids are removed relative to SEQ ID NO. 3;
the calmodulin (CaM) polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO. 4, wherein the fragment has the amino acid sequence of SEQ ID NO: 4 wherein 1-5 amino acids are removed relative to SEQ ID NO. 4; and
the M13 polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO. 5, wherein the fragment has the amino acid sequence of SEQ ID NO: 5 wherein 1-5 amino acids are removed relative to SEQ ID NO. 5.

### Claim 18 (depends on 1)

The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 12.

### Claim 19 (depends on 1)

The isolated polypeptide of claim 1, wherein
the EosFP polypeptide comprises
a C-terminus portion comprising a variant of the amino acid sequence of SEQ ID NO. 2, having at least 98% identity to SEQ ID NO. 2 and
a N-terminus portion comprising a variant of the amino acid sequence of SEQ ID NO. 3, having at least 98% identity to SEQ ID NO. 3;
the calmodulin (CaM) polypeptide comprises a variant of the amino acid sequence of SEQ ID NO. 4, having at least 98% identity to SEQ ID NO. 4; and
the M13 polypeptide comprises a variant of the amino acid sequence of SEQ ID NO. 5, having at least 98% identity to SEQ ID NO. 5.

### Claim 20 (depends on 1)

The isolated polypeptide of claim 1, wherein the EosFP polypeptide includes a C-terminus portion comprising the amino acid sequence of SEQ ID NO. 2 and a N-terminus portion comprising the amino acid sequence of SEQ ID NO. 3; the calmodulin (CaM) polypeptide comprises the amino acid sequence of SEQ ID NO. 4; and the M13 polypeptide comprises a variant of the amino acid sequence of SEQ ID NO. 5, wherein mutations of the variant are selected from the group consisting of S2L, W6Y, W6L, W6V, W6M, W6H, W6F, T9A, T9D, G10D, G10A, H11K, V13H, V13S, V13T, V13A, V13D, V13L.

### Claim 21 (depends on 1)

The isolated polypeptide of claim 1, wherein the isolated polypeptide comprises a variant of the amino acid sequence of SEQ ID NO. 1, having 95% identity to SEQ ID NO: 1.