Abstract
In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms.
Highlights
To date, various bioreactor systems have been established for the production of recombinant proteins
Based on the results of in silico analysis showing that medaka Vtg contains the secretory signal peptide (1MRGLILALSLALVAANQ17) and the receptor-binding region (178HLSKTKDL185), we predicted that the 300-aa N-terminal portion of Vtg (“Vtg signal”) was sufficient to transport the foreign proteins produced in the liver into eggs
The fertilized eggs spawned by both transgenic lines showed the fluorescence of enhanced green fluorescent protein (EGFP) and the luminescence of LUC, respectively, indicating that the Vtg signal-fused proteins were incorporated into the eggs through the Vtg receptor on the surface of the oocytes (Figs. 1c, d, 2c)
Summary
Various bioreactor systems have been established for the production of recombinant proteins. Bacteria can be extensively used owing to the ease of culturing at any scale, but they cannot effectively produce glycosylated proteins of vertebrates, and often form misfolded recombinant proteins as the inclusion body (Villaverde and Carrió 2003). Yeasts and insect cell lines such as Sf9 can perform post-translational modifications (PTMs) to enhance the capability of proteins, it is difficult to completely reproduce the glycosylation pattern of vertebrates; the bioactivities of the produced recombinant proteins may not fully correspond to those of the native forms in vertebrates (Brooks 2004). Mammalian cultured cell lines such as 293T can achieve complex PTMs; culture of the cell lines is so expensive that the recombinant protein cannot be produced cost-effectively. As each technique has both advantages and disadvantages, it is necessary to select the appropriate system depending on the properties of the recombinant proteins
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