Siberian Federal University

Ctenophores are among the oldest and most abundant animals on the planet. In recent years, the range of ctenophores has expanded due to their introduction by cargo ships into the waters of the Black and Baltic Seas. The ctenophore is not a jellyfish, despite some external resemblance to these animals. Ctenophores are quite primitive. These are light transparent creatures with rows of comb-like plates, which act like swimming gear. They lack a heart and a skeleton, but have a nervous system and an amazing ability to produce light, i.e. bioluminescence.

The bioluminescence of ctenophores is accounted for the work of special photosensitive proteins — photoproteins. In the 70s of the 20th century, American scientists William W. Ward and Howard H. Seliger were the first to study a natural photoprotein — berovin, isolated directly from the ctenophore. However, there is no need for using animals for research today. Krasnoyarsk biophysicists have studied an artificially obtained recombinant version of the protein for better understanding its properties.

“Currently, among the Ca2+-regulated coelenterazine-dependent photoproteins, we have cloned four proteins (berovin, bolinopsin, mnemiopsin, and batocyrovin) from different ctenophore species. All of them are responsible for bioluminescence. In bioluminescent organisms, the living light genes are expressed in protein in special cells — photocytes, which, in turn, are assembled into organs of luminescence — photophores. Photoproteins can be used as marker molecules after their production in the form of recombinant proteins. For example, they can be used to visualize various processes in living organisms or in individual tissues in a laboratory,” said Liudmila Burakova, researcher at SibFU Laboratory of Bioluminescent Biotechnologies.

To quantify the light signal and achieve optimal brightness of living light for highlighting animal or human cells with berovin molecules, it is essential to know the quantum yield of the bioluminescent reaction. This indicator is individual for each type of protein. The Krasnoyarsk biophysicists focused on the determination of the quantum yield of berovin. It was the first study conducted in Russia for recombinant photoproteins of ctenophores. Although berovin was cloned in the early 2000s, its operation has not yet been sufficiently studied.

“We obtained highly purified recombinant berovin, quantified its amino acid composition, accurately calculated the concentration required for luminescence, measured its bioluminescent activity, and determined the quantum yield of the reaction. In addition, our team calculated how strongly this protein absorbs light of a given wavelength. We also learned the optimal conditions of luminescence relative to the acid-base balance (pH) and the linear range of the bioluminescent signal when diluting protein in different concentrations,” specified Liudmila Burakova.

Aequorin was the first recombinant photoprotein for which similar calculations were made. It was isolated from the Aequorea victoria jellyfish in the early 1960s and is considered the standard for all known photoproteins. The most accurate quantum yield of aequorin was determined by the Japanese scientist Satoshi Inoue. The Krasnoyarsk biophysicists compared berovin with aequorin to replenish the collection of fundamental knowledge. Surprisingly, these proteins, which are generally characteristic of marine animals, have only functional similarity and are absolutely dissimilar either in amino acid composition or in the way they coordinate the substrate for luminescence.

Finally, the scientists found out that the quantum yield of berovin is lower than that of photoproteins of hydromedusas (in particular, 2.5 less than that of aequorin), and the specific activity of the bioluminescence begins to decrease when the protein is diluted after reaching a concentration of 10 mg/ml. It means that, despite the high analytical potential of ctenophore photoproteins, it might be necessary to change the properties of berovin in the future. For example, one can introduce certain amino acids into its composition to replace existing ones, or use synthetic analogues of the substrate. According to SibFU scientists, it is necessary to study the basic characteristics of protein molecule of berovin profoundly.