Genetically engineered obelin as a bioluminescent label in an assay for a peptide

The marine polyp Obelia longissima produces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of c alcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation of the chromophore, coelenterazine, to coelentera mide with the release of light and CO2. Photoproteins are attractive as lab els in analytical applications because the bioluminescent signal that they produce is the result of a chemical reaction and, therefore, has virtually no background. Thus, bioluminescence allows for extremely sensitive detecti on. In that regard, the feasibility of using obelin as a label has been exp lored with the development of a competitive immunoassay for the determinati on of a small peptide analyte. To attach the obelin label in a controlled m anner to the octapeptide, a fusion protein was produced using recombinant D NA techniques. The protein consisted of the C-terminus of the peptide fused to the N-terminus of obelin. The octapeptide-obelin fusion protein retaine d the bioluminescence properties of the native protein, and was subsequentl y used to generate dose-response curves for the free octapeptide. (C) 1999 Academic Press.