Fluorescent protein-based calcium integrators

Patent metadata

Field	Value
Publication number	US-9518996-B2
Application number	US-201414452428-A
Country	US
Kind code	B2
Filing date	Aug 5, 2014
Priority date	Aug 5, 2013
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

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.

First claim

What is claimed is:

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.

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

3. 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.

4. 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.

5. 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.

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

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

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

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

10. 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.

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

12. 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.

13. 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.

14. 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.

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

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

Assignees

• Hughes Howard Med Inst

Inventors

• Schreiter Eric R
• Looger Loren L
• Fosque Benjamin F

Classifications

• G01N33/52

  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)} · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• G01N33/84 Primary

  involving inorganic compounds or pH · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• C07K14/4728

  Calcium binding proteins, e.g. calmodulin · CPC title