Expression of biologically active red seabream gonadotropins in a baculovirus system

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Recent advances in recombinant gene technology have enabled the production of biologically active fish gonadotropins (GTHs : FSH and LH). However, an efficient expression method for the production of GTHs has not yet been developed. In this study, we attempted to produce red seabream GTHs in silkworm larvae using two different baculovirus expression systems. To synthesize recombinant red seabream heterodimeric GTHs, two strategies were employed. The first utilized a dual vector expression strategy, in which each GTH subunit (α, FSHβ and LHβ) was expressed from a separate comparative vector in a single host. The second method used a bicistronic expression strategy, in which both GTH subunit cDNAs were expressed from the vector. Either a dual or a bicistronic expression vector inoculated into silkworm larvae of the 5th instar early stage. Five days after inoculation, the hemolymph of the larvae containing recombinant FSH and LH was collected, and examined of biochemical property and biological activity. Western blot analysis revealed that by using a dual vector expression system, each subunit of FSH and LH was successfully synthesized and secreted into the hemolymph, allowing the synthesis of heterodimeric molecules. In the case of the bicistronic vector coexpressing GTH subunits, the yields of FSHα and LHα subunits was very low, whereas those of both FSHβ and LHβ subunits were high. The biological activities of GTHs produced by a dual vector system were comparable to those of native GTHs in in vitro steroid hormone production. Both recombinant FSH and LH stimulated the in vitro 11-ketotestosterone production in sliced testis of red seabream. Furthermore, recombinant LH stimulated the in vitro production of estradiol-17β by ovarian follicles in a concentration-dependent manner. Conversely, GTHs derived from a bicistronic vector system had no effect on in vitro steroid hormone production.