Production of recombinant orange-spotted grouper (Epinephelus coioides) luteinizing hormone in insect cells by the baculovirus expression system and its biological effect.
ABSTRACT The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E2) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.
Article: Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems.[show abstract] [hide abstract]
ABSTRACT: Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).Biology of Reproduction 02/2011; 84(6):1171-81. · 4.01 Impact Factor