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US 10,131,960 B2Granted
Genetically encoded fluorescent sensors for detecting intracellular signalling through diacylglycerol pathways
AssigneeMontana Molecular LLC
InventorsThomas E. Hughes, Paul H. Tewson, Anne Marie Quinn
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Abstract
Described herein are novel fluorescent sensors for Diacyl Glycerol (DAG) and phosphatidylinositol 4,5-bisphosphate (PIP2) that are based on circularly permuted fluorescent proteins. These sensors use less visible spectrum than FRET-based sensors, produce robust changes in fluorescence, and can be combined with one another, or with other sensors, in a multiplex assay on standard fluorescent plate readers or live cell imaging systems.
Claims18
A nucleic acid molecule comprising an expression control sequence operably linked to a nucleotide sequence encoding a protein comprising
- a. a protein kinase C (PKC) protein comprising a DAG binding domain and a fusion region;
- and,
- b. a circularly permuted fluorescent protein,
- wherein the fusion region is located between the pseudo substrate domain and the DAG binding domain, or within the DAG binding domain;
- wherein the fluorescent protein is fused with the PKC protein at a fusion site present within the fusion region;
- and,
- wherein the fluorescence of the DAG sensor fusion protein changes upon binding to DAG.
4 more elements
Dependencies are inferred from the claim text.