Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin
Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin
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DOI：
10.1299/jbse.3.221
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发表时间：
2008
影响因子：
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通讯作者：
Koji Iida;M. Murakoshi;S. Kumano;K. Tsumoto;K. Ikeda;Toshimitsu Kobayashi;I. Kumagai;H. Wada
中科院分区：
文献类型：
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作者：
Koji Iida;M. Murakoshi;S. Kumano;K. Tsumoto;K. Ikeda;Toshimitsu Kobayashi;I. Kumagai;H. Wada
普雷斯廷被认为是耳蜗外毛细胞的运动蛋白。由于普雷斯廷的构象变化，OHC被认为是收缩和伸长的，这种OHC运动实现了哺乳动物听觉系统的高灵敏度、宽动态范围和尖锐调谐。自2000年发现以来，普雷斯廷一直受到广泛的研究。因此，通过使用prestin表达细胞的研究，逐渐积累了关于普雷斯廷的结构和功能的知识。普雷斯廷的纯化将允许进一步分析，例如，晶体结构分析，在分子水平上获得关于普雷斯廷的知识。最近，据报道，重组普雷斯廷是从Sf 9昆虫细胞纯化，并进行结构分析，通过电子显微镜。在本研究中，尝试从另一种表达系统，即，用沙鼠普雷斯廷稳定转染的哺乳动物中国仓鼠卵巢（CHO）细胞。首先，由于不清楚哪种去污剂适合于增溶普雷斯廷，因此从通常用于膜蛋白分离的8种去污剂中选择最佳增溶去污剂。还确定了洗涤剂的最佳浓度。结果，明确了10 mM n-壬基-β-D-硫代吡喃麦芽糖苷有效地溶解了普雷斯廷。接着，使用该去污剂，通过抗FLAG亲和层析进行普雷斯廷的纯化，从2 × 109个3 × FLAG标记的表达普雷斯丁的CHO细胞中获得84 ± 23 μ g纯化的普雷斯廷。
Prestin is regarded as the motor protein of cochlear outer hair cells (OHCs). Due to the conformational change of prestin, OHCs are believed to contract and elongate, this OHC motility realizing the high sensitivity, wide dynamic range and sharp tuning of the auditory system of mammals. Since its identification in 2000, prestin has been intensively investigated. As a result, knowledge about the structure and function of prestin has been gradually accumulated by studies using prestin-expressing cells. Purification of prestin would allow further analysis, e.g., crystal structure analysis, to obtain knowledge about prestin at the molecular level. Recently, it has been reported that recombinant prestin was purified from Sf9 insect cells and that structural analysis was carried out by electron microscopy. In the present study, an attempt was made to purify prestin from another expression system, i.e., mammalian Chinese hamster ovary (CHO) cells stably transfected with gerbil prestin. First, since it is unclear which detergents are suitable for solubilization of prestin, the best detergent for solubilization was selected from among 8 kinds of detergent commonly used for membrane protein isolation. The optimum concentration of the detergent was also determined. As a result, it was clarified that 10 mM n-nonyl-β-D-thiomaltopyranoside efficiently solubilizes prestin. Next, using this detergent, purification of prestin by anti-FLAG affinity chromatography was performed, and 84 ± 23 μg of purified prestin was obtained from 2×109 3×FLAG-tagged prestin-expressing CHO cells.