US 9,518,996 B2Granted

Fluorescent protein-based calcium integrators

AssigneeHoward Hughes Medical Institute

InventorsEric R. Schreiter, Loren L. Looger, Benjamin F. Fosque

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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.

Claims21

Dependent claims

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.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/452,428Filed 5 Aug 2014 · us provisional application
Priority: 5 Aug 2013
Published: US 2015/0037812 A15 Feb 2015
Granted: 13 Dec 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

G01N 33/84
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing:…
C07K 14/4728
Not used: calcium binding proteins, e.g. calmodulin
C12N 9/12
Transferases: transferring phosphorus containing groups, e.g. kinases
G01N 33/52
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing: use of…

Family

Application61/862,436Us provisional application · filed 5 Aug 2013
US 2015/0037812 A1Pre-grant publication · published 5 Feb 2015
US 9,518,996 B2This patent

From the US filing history. Foreign family members aren’t listed.

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