Stable mammalian expression of His-tagged prestin in Chinese hamster ovary cells.

Overview

Authors: Yasunori Donjo^1,2, Hisashi Sugimoto¹, Ryosei Motoo^1,2, Manabu Inaba^1,2, Tomokazu Yoshizaki¹, Michio Murakoshi²

ORCID iDs: Michio Murakoshi

Department of Otolaryngology-Head and Neck Surgery, Kanazawa University, Kanazawa, Japan
Faculty of Frontier Engineering, Institute of Science and Engineering, Kanazawa University, Kanazawa, Japan

Institutions: Kanazawa University (Japan)

Journal: Cytotechnology, volume 78, issue 3, article 83

Dates: received 13 February 2026; accepted 1 April 2026; published online 10 April 2026; in print June 2026

Type: Research article · Language: English

License: CC BY

Identifiers: DOI 10.1007/s10616-026-00950-8 · PMID 41969399 · PMCID PMC13069067 · OpenAlex W7153022826

Open access: hybrid, a free copy (OpenAlex)

Status: data only (how the code is found and verified)

Categories: histology / microscopy (modality), other (organism), cellular / molecular (subfield)

Methods: Statistics, fMRI & imaging

Keywords: CHO cells, Stable expression, Membrane protein production, His tag, Prestin

Topic: Hearing, Cochlea, Tinnitus, Genetics (Sensory Systems, Neuroscience), according to OpenAlex

Funding: Kanazawa University

Citations: not cited yet (Europe PMC); 36 references in the paper

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/s10616-026-00950-8.

Reproduced under the paper's license (CC BY), from the paper cited above.

Code

The paper links to its data and to no code of its authors: see the Data section.

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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://doi.org/10.6084/m9.figshare.30013903, https://doi.org/10.6084/m9.figshare.30014410 and https://doi.org/10.6084/m9.figshare.30014431.

Reproduced under the paper's license (CC BY), from the paper cited above.

Versions

The history of this record: each version stored by the harvester or made by a correction of its authors or of the maintainers of its code, and what changed in its facts. The texts of the paper (its abstract, its availability statements) are not part of it; versions that changed only those are not listed.

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://doi.org/10.1007/s10616-026-00950-8

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/s10616-026-00950-8}, url = {https://doi.org/10.1007/s10616-026-00950-8}, 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 - 2026/04/10 VL - 78 IS - 3 SP - 83 SN - 0920-9069 PB - Springer DO - 10.1007/s10616-026-00950-8 UR - https://doi.org/10.1007/s10616-026-00950-8 LA - en ER -

CSL-JSON

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