Production of recombinant eCG with a potent FSH-like activity

马绒毛膜促性腺激素（eCG）由高度糖基化的α和β亚基组成，属于糖蛋白激素家族的成员，该家族包括黄体生成素（LH）和促卵泡激素（FSH）。eCG是促性腺激素家族的独特成员，因为它在马以外的其他物种中引起FSH和LH的反应特征。异源二聚体的正确构象对于有效分泌、激素特异性翻译后修饰、受体结合和信号转导也很重要。作为制备具有强效fsh样活性的重组eCG的途径，我们分析了n -连接寡糖在eCG生物活性中的作用，发现n -连接寡糖的去除对eCG的fsh样活性具有抗性。我们还研究了α亚基和β亚基是否可以合成为具有任何生物活性的单一多肽链（栓系eCG），使用拴系eCG分子，其中eCG β亚基的羧基端与α亚基的氨基端融合。瞬时转染的CHO-K1细胞有效地分泌拴系心电图，并表现出与二聚体心电图相似的lh样活性。有趣的是，与原生型和野生型eCG相比，系留型eCG的fsh样活性明显增加。我们的数据首次表明，拴系ecg可以在CHO-K1细胞中有效表达，而不会失去lh和fsh样活性。我们的研究结果还表明，具有强效fsh样活性的重组eCG蛋白可能由大肠杆菌产生，其中很少发生糖基化。

Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha-and beta-subunits and belongs to the member of the glycoprotein hormone family which includes luteinizing hormone (LH) and follicle-stimulating hormone (FSH). eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in others species than the horse. The correct conformation of the heterodimer is also important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduction. As an approach to produce recombinant eCG with a potent FSH-like activity, we analyzed the roles of N-linked oligosaccharides in the biological activities of eCG and found that the FSH-like activity of eCG is resistant to the removal of N-linked oligosaccharides. We also investigated whether alpha and beta subunits can be synthesized as a single polypeptide chain (tethered-eCG) with any biological activities by using the tethered-eCG molecule in which the carboxyl terminus of the eCG beta-subunit was fused to the amino terminus of the alpha-subunit. The tethered-eCG was efficiently secreted from the transiently transfected CHO-K1 cells and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. Our data for the first time suggest that the tethered-eCG can be expressed efficiently in the CHO-K1 cells without losing LH-and FSH-like activities. Our results also suggest that the recombinant eCG protein with a potent FSH-like activity can possibly be pruduced by E. coli in which very little of the glycosylation occurs.