Carbon-Neutral Resources Research Center, Hankyong National University, Anseong 17579, Republic of Korea.
Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Republic of Korea.
Int J Mol Sci. 2024 Jul 2;25(13):7282. doi: 10.3390/ijms25137282.
This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115-149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.
本研究旨在制备具有高活性的单链重组鳗鲡滤泡刺激素（rec-eel FSH）类似物，该类似物在仓鼠卵巢 DG44（CHO DG44）细胞中具有高活性。我们最近报道，马绒毛膜促性腺激素（eCG）β亚基的 O-连接糖基化羧基末端肽（CTP）有助于在哺乳动物细胞中发挥高活性和时间依赖性分泌。我们构建了一个突变体（FSH-M），其中包含 eCG β亚基 CTP 区域（氨基酸 115-149）的连接子被插入到野生型单链鳗鲡 FSH（FSH-wt）的β亚基和α亚基之间。含有鳗鲡 FSH-wt 和鳗鲡 FSH-M 的质粒被转染到 CHO DG44 细胞中，从 10 和 7 个克隆中分离出表达每种蛋白质的单个细胞。在培养期间，分泌逐渐增加，在第 9 天达到 4000-5000ng/mL 的峰值。eel FSH-wt 的分子量为 34-40 kDa，而 eel FSH-M 的分子量显著增加，有两个 39-46 kDa 的条带。用 PNGase F 处理以去除糖基化位点会使分子量显著降低至约 8 kDa。eel FSH-M 的 EC 值和最大反应性大约比 eel FSH-wt 高 1.23-和 1.06 倍，表明突变体显示出略高的生物学活性。磷酸化细胞外调节激酶（pERK1/2）激活在 5 分钟时出现急剧峰值，随后迅速下降。这些发现表明，具有 eCG β亚基 CTP 接头的新型 rec-eel FSH 分子具有强大的活性，并且可以使用稳定的 CHO DG44 细胞系统大量生产。
