Expression, Purification, and Determination of Sensitivity to Calcium Ions of Ctenophore Photoproteins

Light-sensitive Ca²⁺-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca²⁺-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence. However, in contrast to the related Ca²⁺-regulated photoproteins of jellyfish their capacity to bioluminescence disappears on exposure to light over the entire absorption spectral range of ctenophore photoproteins. Here, we describe protocols for expression of gene encoding ctenophore photoprotein in Escherichia coli cells, obtaining of the recombinant apoprotein of high purity and its conversion into active photoprotein with synthetic coelenterazine as well as determination of its sensitivity to calcium ions using light-sensitive Ca²⁺-regulated photoprotein berovin from ctenophore Beroe abyssicola as an illustrative case.