Cloning and sequence analysis of cDNA for the luminescent protein aequorin

Parts used

Key findings

• Inouye et al. constructed a cDNA library from Aequorea victoria comprising ~1.5×10⁴ independent clones and screened it by colony hybridization using two synthetic oligonucleotide probes. Three positive clones were isolated; clone AQ440 was fully sequenced by the Maxam-Gilbert method and contained a complete open reading frame encoding apoaequorin.
• The deduced amino acid sequence established apoaequorin as a 189-residue protein (MW 21,400) with three E-F hand Ca²⁺-binding structures. Sequence comparison with bovine calmodulin revealed high homology at all three Ca²⁺-binding sites, and chemical amino acid composition analysis confirmed consistency with the nucleotide-derived sequence.
• Hydropathy profiling identified three hydrophobic regions at positions 40–50, 55–65, and 100–110 as candidate interaction sites for the chromophore coelenterazine. Three cysteine residues at positions 145, 152, and 180 near the third E-F hand were implicated in Ca²⁺-triggered luminescence; SH-blocking by N-ethylmaleimide abolished activity and 2-mercaptoethanol was required for aequorin regeneration.

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 E-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.