Motoo Ryosei, Sugimoto Hisashi, Donjo Yasunori, Yoshizaki Tomokazu, Murakoshi Michio
Department of Otolaryngology-Head and Neck Surgery, Kanazawa University, Kanazawa, Japan.
Faculty of Frontier Engineering, Institute of Science and Engineering, Kanazawa University, Kanazawa, Japan.
PLoS One. 2024 Dec 2;19(12):e0314517. doi: 10.1371/journal.pone.0314517. eCollection 2024.
The electromotility of outer hair cell is considered to be based on voltage-dependent conformational changes in the motor protein prestin. The structure and function of prestin have been increasingly examined in recent years. To obtain further information on prestin, a method to stably obtain prestin as the material for this research is required. This study attempted to construct a stable expression system for prestin on Chinese hamster ovary (CHO) cells and its function was evaluated. His-tagged prestin expression vectors were transfected into CHO cells and high-expression clones were obtained by drug selection and limiting dilution method. The expression and activity of prestin were examined by Western blotting and immunofluorescent experiments and by patch clamping. Clones with high fluorescent intensity tended to exhibit bell-shaped non-linear capacitance, suggesting the active function of prestin in such clones. The stable expression and activity of prestin in CHO cells were confirmed in 7 clones out of twelve clones developed. Although expression levels were 1/60-1/30 of those in OHCs, cell line maintaining the stable expression of functional prestin with His tag will be advantageous for future analyses of its structure and function.
外毛细胞的电运动性被认为基于马达蛋白prestin中电压依赖性的构象变化。近年来，prestin的结构和功能受到了越来越多的研究。为了获得关于prestin的更多信息，需要一种稳定获取prestin作为本研究材料的方法。本研究试图构建一个在中华仓鼠卵巢（CHO）细胞上稳定表达prestin的系统，并对其功能进行评估。将带有His标签的prestin表达载体转染到CHO细胞中，通过药物筛选和有限稀释法获得高表达克隆。通过蛋白质免疫印迹法、免疫荧光实验和膜片钳技术检测prestin的表达和活性。荧光强度高的克隆倾向于表现出钟形的非线性电容，表明这些克隆中prestin具有活性功能。在所培养的12个克隆中，有7个克隆证实了prestin在CHO细胞中的稳定表达和活性。尽管表达水平仅为外毛细胞中的1/60 - 1/30，但维持带有His标签的功能性prestin稳定表达的细胞系将有利于未来对其结构和功能的分析。
