Production and accumulation of recombinant protein via vitellogenin transport pathway in Daphnia magna

The demand for recombinant proteins is rising and various systems for protein production have been developed, including cultured cells, yeast, and bacteria. This study explores the use of Daphnia, a small crustacean, as a host for recombinant protein production. Daphnia can be cultured easily and inexpensively. We aimed to accumulate recombinant proteins in its eggs. This research aimed to identify the signal peptides necessary for transporting vitellogenin into Daphnia eggs and to develop a system for transporting recombinant proteins into the eggs using these signals. A fusion protein of GFP (Green Fluorescent Protein) with a predicted transport signal sequence was expressed in D. magna, and we confirmed that GFP is secreted into the hemolymph. Additionally, a specific region of about 300 amino acids, presumed necessary for binding to vitellogenin receptors, was added to the fusion GFP and expressed outside the ovaries, resulting in strong GFP fluorescence in the eggs. These results indicate that by fusing signals for protein secretion and those that enable uptake into eggs, recombinant protein can be encapsulated into the eggs. This system shows promise as a simple and cost-effective method for producing recombinant proteins by accumulating them in Daphnia eggs.