Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells.
The electromotility of outer hair cell is considered to be based on voltage-dependent conformational changes in the motor protein prestin. The structure and function of prestin have been increasingly examined in recent years. To obtain further information on prestin, a method to stably obtain presti...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2024-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0314517 |
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| author | Ryosei Motoo Hisashi Sugimoto Yasunori Donjo Tomokazu Yoshizaki Michio Murakoshi |
| author_facet | Ryosei Motoo Hisashi Sugimoto Yasunori Donjo Tomokazu Yoshizaki Michio Murakoshi |
| author_sort | Ryosei Motoo |
| collection | DOAJ |
| description | The electromotility of outer hair cell is considered to be based on voltage-dependent conformational changes in the motor protein prestin. The structure and function of prestin have been increasingly examined in recent years. To obtain further information on prestin, a method to stably obtain prestin as the material for this research is required. This study attempted to construct a stable expression system for prestin on Chinese hamster ovary (CHO) cells and its function was evaluated. His-tagged prestin expression vectors were transfected into CHO cells and high-expression clones were obtained by drug selection and limiting dilution method. The expression and activity of prestin were examined by Western blotting and immunofluorescent experiments and by patch clamping. Clones with high fluorescent intensity tended to exhibit bell-shaped non-linear capacitance, suggesting the active function of prestin in such clones. The stable expression and activity of prestin in CHO cells were confirmed in 7 clones out of twelve clones developed. Although expression levels were 1/60-1/30 of those in OHCs, cell line maintaining the stable expression of functional prestin with His tag will be advantageous for future analyses of its structure and function. |
| format | Article |
| id | doaj-art-ebd64d1d248c4513bfcad8717bdbd533 |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-ebd64d1d248c4513bfcad8717bdbd5332024-12-06T05:31:12ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-011912e031451710.1371/journal.pone.0314517Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells.Ryosei MotooHisashi SugimotoYasunori DonjoTomokazu YoshizakiMichio MurakoshiThe electromotility of outer hair cell is considered to be based on voltage-dependent conformational changes in the motor protein prestin. The structure and function of prestin have been increasingly examined in recent years. To obtain further information on prestin, a method to stably obtain prestin as the material for this research is required. This study attempted to construct a stable expression system for prestin on Chinese hamster ovary (CHO) cells and its function was evaluated. His-tagged prestin expression vectors were transfected into CHO cells and high-expression clones were obtained by drug selection and limiting dilution method. The expression and activity of prestin were examined by Western blotting and immunofluorescent experiments and by patch clamping. Clones with high fluorescent intensity tended to exhibit bell-shaped non-linear capacitance, suggesting the active function of prestin in such clones. The stable expression and activity of prestin in CHO cells were confirmed in 7 clones out of twelve clones developed. Although expression levels were 1/60-1/30 of those in OHCs, cell line maintaining the stable expression of functional prestin with His tag will be advantageous for future analyses of its structure and function.https://doi.org/10.1371/journal.pone.0314517 |
| spellingShingle | Ryosei Motoo Hisashi Sugimoto Yasunori Donjo Tomokazu Yoshizaki Michio Murakoshi Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells. PLoS ONE |
| title | Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells. |
| title_full | Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells. |
| title_fullStr | Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells. |
| title_full_unstemmed | Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells. |
| title_short | Expression and functional characterization of His-tagged prestin in Chinese hamster ovary cells. |
| title_sort | expression and functional characterization of his tagged prestin in chinese hamster ovary cells |
| url | https://doi.org/10.1371/journal.pone.0314517 |
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