Cloning and sequence analysis of cDNA for the luminescent protein aequorin.

Abstract

The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic oligonucleotide probes. The primary structure of the protein deduced from the nucleotide sequence showed that the protein is composed of 189 amino acid residues and has three F-F hand structures that are characteristic for Ca2+-binding sites. The sequence also suggested that the protein has hydrophobic regions at which the protein may interact with its functional chromophore, coelenterazine.

A phogrin–aequorin chimaera to image free Ca2+ in the vicinity of secretory granules

“…To avoid cleavage of the internal EcoRV site within the aequorin gene-coding region [9], the 618 bp EcoRV-XbaI fragment was subcloned into the intermediate vector pGEM-T (Promega) and released by partial digestion with XbaI and EcoRV. cDNA encoding the entire coding region of phogrin within the eukaryotic expression vector pcDNA3 (Invitrogen) was cleaved with EcoRV and XbaI, and the HAaequorin fragment inserted to generate plasmid pCMV.phog.Aq (Figure 1).…”

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

“…1,2) Aequorin is a stable complex of apoprotein (apoaequorin, 21.4 kDa protein) and 2-peroxycoelenterazine, 3) and emits light ( max at $470 nm) when bound to Ca 2þ ions (>10 À7 M). 1,4) The luminescence reaction of aequorin triggered by Ca 2þ is fairly fast (within a few seconds), and the luminescence signal shows a high signal-tonoise ratio due to a low luminescence background.…”

Prix Nobel de Chimie 2008 (Osumo Shimomura, Martin Chalfie et Roger Y. Tsien) : Osamu Shimomura

“…Cependant, l'aequorine émet peu de photons et il fallut attendre 1978 pour effectuer les première expériences d'imagerie calcique [9]. L'aequorine a été clonée en 1985 simultanément par deux groupes [10,11] …”