Journal of Biomechanical Science and Engineering
Online ISSN : 1880-9863
ISSN-L : 1880-9863
Papers
Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin
Koji IIDAMichio MURAKOSHIShun KUMANOKouhei TSUMOTOKatsuhisa IKEDAToshimitsu KOBAYASHIIzumi KUMAGAIHiroshi WADA
Author information
JOURNALS FREE ACCESS

2008 Volume 3 Issue 2 Pages 221-234

Details
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 2×109 3×FLAG-tagged prestin-expressing CHO cells.

Information related to the author
© 2008 by The Japan Society of Mechanical Engineers
Previous article Next article
feedback
Top