Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal, Consejo Superior de Investigaciones Cientiíficas, Ribera de Cabanes, Castellón, Spain.
Biol Reprod. 2011 Jun;84(6):1171-81. doi: 10.1095/biolreprod.110.086470. Epub 2011 Feb 3.
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).
不同的生产系统和 DNA 构建物会导致重组尖吻鲈促性腺激素产生不同的产率、生物效价和体内药代动力学，但它们都能特异性地激活相应的受体。促性腺激素（GTHs）是糖蛋白激素，在调节性腺功能方面发挥着重要作用。最近，我们成功地从尖吻鲈脑垂体中分离出天然的尖吻鲈 Fsh，但为了确保生物活性 GTH 的可用性且不与其他相关糖蛋白发生交叉污染，我们使用昆虫和哺乳动物细胞以及不同的构建物来生产重组尖吻鲈 GTH，这些构建物产生的是连接或非共价结合的二聚体。我们研究并比较了它们的产量、对同源受体的结合特异性以及生物活性。这两种表达系统都成功地生成了具有生物活性的重组 GTH，但昆虫 Sf9 细胞产生的重组蛋白量高于哺乳动物中国仓鼠卵巢（CHO）稳定克隆。所有重组 GTH 都能激活其同源受体，而不会发生交叉配体结合，并且能够在体外刺激尖吻鲈性腺类固醇生成，尽管生物效价不同。为了评估它们在体内应用中的效果，我们评估了它们在尖吻鲈血浆中的半衰期。与 CHO-GTHs 相比，Sf9-GTHs 的体内稳定性较低，因为它们在血液循环中被迅速清除。细胞依赖性糖基化可能是影响这些重组糖蛋白最终体内稳定性和生物效价的原因。总之，昆虫和哺乳动物表达系统都能产生具有生物活性的重组尖吻鲈促性腺激素，但具有不同的特点，可用于不同的目的（例如，分别用于抗体生产或体内研究）。
