Construction of an expression system for the motor protein prestin in Chinese hamster ovary cells.
The electromotility of outer hair cells (OHCs) is believed to be a major factor in cochlear amplification that enables the high sensitivity of hearing in mammals. This motility is thought to be based on voltage-dependent conformational changes of a motor protein embedded in the lateral wall of the OHC. In 2000, this motor protein was identified and termed prestin. To obtain knowledge on the function of prestin, research at the molecular level is necessary. For this purpose, a method of obtaining a large amount of prestin is required. In this study, an attempt was therefore made to construct an expression system for prestin. Prestin cDNA was introduced into Escherichia coli (E. coli), insect cells and Chinese hamster ovary (CHO) cells, and the expression of prestin was examined by Western blotting. As CHO cells expressed prestin well, we generated prestin-expressing cell lines using CHO cells by limiting dilution cloning. The stable expression and the activity of prestin in generated cell lines were then confirmed. Finally, to obtain prestin from these cell lines efficiently, culture conditions of the cells were examined, and it was clarified that cells should be cultured in serum-free medium and harvested around 48 h after passage.
Iida
Koji
K
Department of Bioengineering and Robotics, Tohoku University, 6-6-01 Aoba-yama, Sendai 980-8579, Japan.
Tsumoto
Kouhei
K
Ikeda
Katsuhisa
K
Kumagai
Izumi
I
Kobayashi
Toshimitsu
T
Wada
Hiroshi
H
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D004594:Electrophysiology; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D018076:DNA, Complementary; C498722:Pres protein, Cavia porcellus; D011506:Proteins; D011994:Recombinant Proteins