{"title":"Stable mammalian expression of His-tagged prestin in Chinese hamster ovary cells.","authors":"Yasunori Donjo, Hisashi Sugimoto, Ryosei Motoo, Manabu Inaba, Tomokazu Yoshizaki, Michio Murakoshi","doi":"10.1007/s10616-026-00950-8","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1007/s10616-026-00950-8.</p>","PeriodicalId":10890,"journal":{"name":"Cytotechnology","volume":"78 3","pages":"83"},"PeriodicalIF":1.7000,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13069067/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cytotechnology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s10616-026-00950-8","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2026/4/10 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍：
The scope of the Journal includes:
1. The derivation, genetic modification and characterization of cell lines, genetic and phenotypic regulation, control of cellular metabolism, cell physiology and biochemistry related to cell function, performance and expression of cell products.
2. Cell culture techniques, substrates, environmental requirements and optimization, cloning, hybridization and molecular biology, including genomic and proteomic tools.
3. Cell culture systems, processes, reactors, scale-up, and industrial production. Descriptions of the design or construction of equipment, media or quality control procedures, that are ancillary to cellular research.
4. The application of animal/human cells in research in the field of stem cell research including maintenance of stemness, differentiation, genetics, and senescence, cancer research, research in immunology, as well as applications in tissue engineering and gene therapy.
5. The use of cell cultures as a substrate for bioassays, biomedical applications and in particular as a replacement for animal models.
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