Stable mammalian expression of His-tagged prestin in Chinese hamster ovary cells.
Overview
- Department of Otolaryngology-Head and Neck Surgery, Kanazawa University, Kanazawa, Japan
- Faculty of Frontier Engineering, Institute of Science and Engineering, Kanazawa University, Kanazawa, Japan
Abstract
Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins, including membrane proteins that require a mammalian cellular environment for proper folding and targeting. Prestin, a motor protein responsible for outer hair cell electromotility in the mammalian cochlea, is a multi-pass membrane protein whose structural and functional analyses require reliable expression systems capable of producing full-length protein. In the present study, we established CHO cell lines stably expressing full-length prestin with a C-terminal 6×histidine (His) tag using mammalian expression vectors driven by either the elongation factor-1α (EF1α) promoter or the cytomegalovirus (CMV) promoter. Following geneticin selection and limiting dilution cloning, multiple clonal cell lines were obtained and characterized by Western blotting, immunofluorescence microscopy and electrophysiological analysis. Seven clones expressing His-tagged prestin were identified. Quantitative Western blot analysis using a calibrated His-tagged reference protein demonstrated that the EF1α-driven system yielded higher-producing clones than the CMV-driven system, with a maximum estimated production of approximately 271 µg per 2 × 10⁹ cells. Immunofluorescence imaging confirmed membrane localization of prestin in expressing clones. Whole-cell patch-clamp recordings revealed nonlinear capacitance, indicating functional activity of the expressed protein. These results demonstrate that CHO cells provide a stable mammalian expression system for the production of full-length His-tagged prestin. The system enables reproducible protein production, quantitative expression evaluation and functional validation within a single cellular background, and may facilitate future biochemical, structural and functional studies of prestin and related membrane proteins.
Supplementary Information: The online version contains supplementary material available at 10.1007/
Reproduced under the paper's license (CC BY), from the paper cited above.
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Data
Datasets cited
- figshare:30013903 — at figshare; found in “Data availability”
- figshare:30014410 — at figshare; found in “Data availability”
- figshare:30014431 — at figshare; found in “Data availability”
Data availability
All datasets are available in the figshare repository at https://
Reproduced under the paper's license (CC BY), from the paper cited above.
Versions
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Version 1, 29 September 2026: the first record
Recorded: type, language, journal, volume, issue, pages, dates, 6 authors, 5 keywords, 1 funder, 31 references.
Cite
This paper
Donjo, Y., Sugimoto, H., Motoo, R., Inaba, M., Yoshizaki, T., & Murakoshi, M. (2026). Stable mammalian expression of His-tagged prestin in Chinese hamster ovary cells. Cytotechnology, 78(3), 83. https://
BibTeX
@article{donjo2026stable
author = {Donjo, Yasunori and Sugimoto, Hisashi and Motoo, Ryosei and Inaba, Manabu and Yoshizaki, Tomokazu and Murakoshi, Michio},
title = {{Stable mammalian expression of His-tagged prestin in Chinese hamster ovary cells}},
journal = {Cytotechnology},
year = {2026},
month = apr,
volume = {78},
number = {3},
pages = {83},
publisher = {Springer},
issn = {0920-9069},
doi = {10.1007/
url = {https://
pmid = {41969399},
pmcid = {PMC13069067}
}
RIS
TY - JOUR
AU - Donjo, Yasunori
AU - Sugimoto, Hisashi
AU - Motoo, Ryosei
AU - Inaba, Manabu
AU - Yoshizaki, Tomokazu
AU - Murakoshi, Michio
TI - Stable mammalian expression of His-tagged prestin in Chinese hamster ovary cells
T2 - Cytotechnology
J2 - Cytotechnology
PY - 2026
DA - 2026/
VL - 78
IS - 3
SP - 83
SN - 0920-9069
PB - Springer
DO - 10.1007/
UR - https://
LA - en
ER -
CSL-JSON
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