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，该家族包括促黄体激素（LH）和促卵泡激素（FSH）。eCG是促性腺激素家族的独特成员，因为它在除马以外的其他物种中激发FSH和LH的反应特征。异二聚体的正确构象对于有效分泌、酶特异性翻译后修饰、受体结合和信号转导也是重要的。作为生产具有有效FSH样活性的重组eCG的方法，我们分析了N-连接寡糖在eCG生物活性中的作用，发现eCG的FSH样活性对N-连接寡糖的去除具有抗性。我们还研究了α和β亚基是否可以通过使用其中eCG β亚基的羧基末端与α亚基的氨基末端融合的栓系eCG分子合成为具有任何生物活性的单一多肽链（栓系eCG）。栓系的eCG有效地从瞬时转染的CHO-K1细胞中分泌，并显示出与二聚体eCG相似的LH样活性。有趣的是，与天然和野生型eCG相比，栓系eCG的FSH样活性显著增加。我们的数据第一次表明，栓系-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，重组eCG蛋白具有较强的FSH样活性，可能会产生的E。在大肠杆菌中几乎没有发生糖基化。

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.