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

  • US 7,202,046 B2
  • Filed: 02/12/2004
  • Issued: 04/10/2007
  • Est. Priority Date: 06/05/1997
  • Status: Expired due to Term
First Claim
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1. A process for detecting and/or quantifying non-covalent interactions between a target receptor and one of its ligands, comprising:

  • preparing cells or cell fragments containing a nucleic acid sequence encoding a fluorescent protein fused with a nucleic acid sequence encoding the target receptor, the fusion between the nucleic acid sequence for the fluorescent protein and the nucleic acid sequence for the target receptor being such that the properties of the target receptor are not modified by the presence of the fluorescent protein;

    wherein the interaction between the target receptor, and the ligand is not modified, andwherein a response transduction function is not modified, the fluorescent protein is selected from Green Fluorescent Proteins (GFPs) obtained or derived from autofluorescent proteins of Cnidarians, the molar extinction coefficient of which is greater than about 14,000 M

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    and the quantum fluorescence yield is greater than about 0.38placing said cells or said cell fragments in contact with a ligand for said target receptor, said ligand labeled with a label capable of absorbing the light emitted by the fluorescent protein, the fluorescent protein being the fluorescence energy donor and the label being the fluorescence energy acceptor, or the fluorescent protein being the fluorescence energy acceptor and the label being a fluorescent substance which is a fluorescence energy donor; and

    irradiating said cells or said cell fragments at a wavelength which makes it possible either to excite the fluorescent protein or to excite the label,wherein the steps of placing in contact and irradiating are carried out either simultaneously or one after the other, orsaid cells or said cell fragments are placed in contact with a ligand for said target receptor said ligand labeled with a label, the cells or the ligand having been irradiated before being placed in contact,wherein a reduction in the amplitude of the donor'"'"'s emission and/or emission signal characteristic of the acceptor'"'"'s emission is measured and measuring the fluorescence energy transfer when quantifying the non-covalent interactions.

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