Fluorescent proteins

An isolated fluorescent protein having chemiluminescence activity, comprising in a complex an apoprotein that is a component of a calcium-binding photoprotein, a coelenteramid or an analog thereof, and a calcium ion or a divalent or trivalent ion that can be substituted for the calcium ion,

• wherein the coelenteraminde or the analog thereof is represented by the following formula (1) or (2):
• wherein
• R1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted arylated alkyl group, or a straight or branched chain alkyl group that may be substituted by an aliphatic cyclic group;
• R2 is a substituted or unsubstituted aryl group, a substituted or unsubstituted arylated alkyl group, a substituted or unsubstituted aryl alkenyl group, a straight or branched chain alkyl group that may be substituted by an aliphatic cyclic group, a straight or branched chain alkenyl group that may be substituted by an aliphatic cyclic group, or a heterocyclic group;
• R3 is a hydrogen atom, a substituted or unsubstituted alkyl group;
• X1 is a hydrogen atom, a hydroxyl group, a halogen atom, an alkoxyl group, or an amino group;
• X2 is a hydrogen atom or a hydroxyl group;
• and
• is a divalent hydrocarbon group having 1 to 4 carbon atoms;
• wherein the coelenteraminde remains coordinated inside the apoprotein,
• wherein the calcium ion or the divalent or trivalent ion that can be substituted for the calcium ion is bound to an EF-hand of the apoprotein, and
• wherein the sulflydryl groups of the cysteine residues of the apoprotein exists without forming a disulfide bond such that the isolated fluorescent protein maintains chemiluminescence activity.

A method for detecting a target substance, comprising:

• a step of binding a ligand for the target substance to a fluorescent protein having chemiluminescence activity to provide a ligand bound fluorescent protein having chemiluminescence activity, wherein the fluorescent protein comprises in a complex an apoprotein that is a component of a calcium-binding photoprotein, a coelenteramide or an analog thereof, and a calcium ion or a divalent or trivalent ion that can be substituted for the calcium ion,
• a step of binding the ligand bound fluorescent protein having chemiluminescence activity to the target substance to be bound via the ligand,
• a step of emitting light by adding a coelenterazine or an analog thereof, and
• a step of detecting the emitted light;
• wherein the coelenteramide remains coordinated inside the apoprotein,
• wherein the calcium ion or the divalent or trivalent ion that can be substituted for the calcium ion is bound to an EF-hand of the apoprotein, and
• wherein the sulfhydryl groups of the cysteine residues of the apoprotein exists without forming a disulfide bond such that the fluorescent protein maintains chemiluminescence activity.

A method for enhancing thermal stability of a fluorescent protein having chemiluminescence activity, wherein the fluorescent protein comprises in a complex an apoprotein that is a component of a calcium-binding photoprotein, a coelenteramide or an analog thereof, and a calcium ion or a divalent or trivalent ion that can be substituted for the calcium ion,

• wherein a reducing agent is added to a solution in which the fluorescent protein is dissolved;
• wherein the coelenteramide remains coordinated inside the apoprotein;
• wherein the calcium ion or the divalent or trivalent ion that can be substituted for the calcium ion is bound to an EF-hand of the apoprotein, and
• wherein the sulflydryl group of the cysteine residues of the apoprotein exists without forming a disulfide bond such that the fluorescent protein maintains chemiluminescence activity.