Patent ID: 7700303

Claim:
A method for preparing a biosensor consisting of (i) at least one fragment of a receptor which is a protein having one or more disulfide bridges, wherein said receptor fragment is capable of binding to a suitable ligand via an active site to form a receptor fragment-ligand complex, and in which receptor fragment at least one of its amino acid residues located in the proximity of said active site is naturally present in the form of a Cys residue, or is substituted with a Cys residue, and (ii) a fluorophore coupled to said Cys residue(s), comprising: (a) selecting residues of the receptor by searching for the residues which, in the receptor-ligand complex, (i) are in direct contact with the ligand, or (ii) are in contact via a water molecule, or (iii) have a solvent accessible surface area (ASA) which is modified by the binding of the ligand, when use is made of spheres of increasing radius of 1.4 to 30 Å, preferably of 1.4 to 2.9 Å, for the molecule of said solvent; (b) calculating the solvent accessible surface area (ASA), for the free receptor, of the atoms in the γ position and, optionally, in the δ position for each residue selected in (a), using a sphere of 1.4 Å, and selecting the residues in which the atom in the γ position or the atom in the δ position is accessible to the solvent; (c) mutating by site-directed mutagenesis at least one of the residues selected in (b) to a Cys residue when said residue is not naturally a Cys residue, and (d) coupling the Sγ atom of at least one Cys residue obtained in (b) or in (c) to a fluorophore.