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

Abstract

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 2×10 9 3×FLAG-tagged prestin-expressing CHO cells.

Atomic force microscopy imaging of the structure of the motor protein prestin reconstituted into an artificial lipid bilayer

“…The purification of prestin was performed by the method established in our previous study with some modifications [5]. CHO cells which had been modified to stably express C‐terminal 3×FLAG‐tagged prestin were suspended in Tris–KCl buffer (10 mM Tris, 150 mM KCl, pH 7.4) and sonicated, followed by centrifugation at 1000× g for 7 min at 4 °C to remove nuclei and undisrupted cells.…”