Use of a fluorescent protein for detecting interactions between a target protein and its ligand

• US 20060275828A1
• Filed: 05/10/2006
• Published: 12/07/2006
• Est. Priority Date: 06/05/1997
• Status: Abandoned Application

• Current Assignee: Centre National De La Recherche Scientifique
• Original Assignee: Centre National De La Recherche Scientifique
• Inventors: Galzi, Jean-Luc, Alix, Philippe
• Application Number:
  US11/430,871
• Publication Number:
  US 20060275828A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: G01N 2333/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

A fluorescent protein selected in particular among the autofluorescent proteins for detecting the non-covalent interaction between a target protein marked by the fluorescent protein and one of its ligands marked by a marker consisting of: either a molecule capable of absorbing the light emitted by the fluorescent protein, or a fluorescent substance, the detection taking place by fluorescence energy transfer: between the fluorescent protein and the fluorescent substance, the fluorescent substance being such that it is excitable at the fluorescent protein emitting wavelength, or it emits at the fluorescent protein emitting wavelength; between the fluorescent protein and the molecule capable of absorbing the light emitted by the fluorescent protein.

Claims

1. A process for identifying and/or quantifying interactions between a receptor and a non-fluorescent molecule which is biologically active with respect to said receptor, wherein said non-fluorescent molecule forms a reversible non-covalent interaction with said receptor, comprising adding a biologically active non-fluorescent molecule to cells, or cell fragments that express DNA coding for a receptor labeled with fluorescent protein and for a labeled G protein, characterized in that:

i) an agonist and biologically active non-fluorescent molecule triggers a signal transduction detected by a variation in the energy transfer between the receptor labeled with the fluorescent protein and the G protein labeled with the label;

or

ii) an antagonistic biologically active non-fluorescent molecule inhibits the signal transduction brought about by an agonist and detected by variation in the transfer of fluorescence energy between the receptor labeled with the fluorescent protein and the G protein labeled with the label.

2. The process according to claim 1, wherein the fluorescent protein is Enhanced Green Fluorescent Protein (EGFP) and the labeled substance is Bodipy and in which either the reduction in the emissions amplitude of EGFP or an emission signal of Bodipy resulting from an energy transfer is detected, the irradiation wavelength corresponding to the excitation wavelength of EGFP.

3. The process according to claim 1, wherein the fluorescent protein is Enhanced Green Fluorescent Protein (EGFP) and the labeled substance is a coumarin, and wherein either the diminution of amplitude of coumarin or an emission signal EGFP resulting from an energy transfer is detected, said irradiation wavelength corresponding to the excitation wavelength of coumarin.

4. The process according to claim 1, wherein the fluorescent protein is fused on the N-terminal side and the target protein is fused on the C-terminal side.

5. The process according to claim 1, wherein the fluorescent protein is fused on the C-terminal side and the target protein is fused on the N-terminal side.

6. The process according to claim 1, wherein the fluorescent protein is inserted into a receptor target protein wherein said receptor is coupled to the G protein, this insertion taking place in the first or third intracellular loop of the receptor, with the proviso that the insertion does not destroy either the properties of the receptor or the fluorescence of the fluorescent protein.

7. The process according to claim 1, wherein the cells are mammalian cells, which are adherent or in suspension selected from the group consisting CHO cells, COS cells, lyphocytic lines, fibroblasts, yeast cells pichia pastoris, saccharomyces cerevisia, saccharomyces kluyveri, Hansenula polymorpha, and insect cells.