Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin

Journal of Biomechanical Science and Engineering

Izumi KUMAGAI¹  Toshimitsu KOBAYASHI²  Shun KUMANO⁵  Hiroshi WADA⁵  Michio MURAKOSHI⁵  Kouhei TSUMOTO⁴  Katsuhisa IKEDA³  Koji IIDA⁵ 

[1] Department of Biomolecular Engineering, Tohoku University;Department of Otorhinolaryngology, Tohoku University, Graduate School of Medicine;Department of Otorhinolaryngology, Juntendo University School of Medicine;Department of Medical Genome Sciences, The University of Tokyo;Department of Bioengineering and Robotics, Tohoku University

关键词: Outer Hair Cell;    Motor Protein Prestin;    Chinese Hamster Ovary Cell;    Membrane Protein;    Detergent;    n-Nonyl-β-D-thiomaltopyranoside;    Solubilization;    Anti-FLAG Affinity Chromatography;    Purification;   

DOI  :  10.1299/jbse.3.221

来源: Japan Society of Mechanical Engineers

【 摘 要 】

References(32)Cited-By(2)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.

【 授权许可】

Unknown