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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Bibliographic Details
Published in:PloS one 2024-12, Vol.19 (12), p.e0314517
Main Authors: Motoo, Ryosei, Sugimoto, Hisashi, Donjo, Yasunori, Yoshizaki, Tomokazu, Murakoshi, Michio
Format: Article
Language:English
Subjects:
Analysis
Animals
Anion Transport Proteins - genetics
Anion Transport Proteins - metabolism
Biology and Life Sciences
Capacitance
CHO Cells
Cloning
Cricetinae
Cricetulus
Dilution
Experiments
Expression vectors
Fluorescence
Gene Expression
Hair cells (Mechanoreceptors)
Hair Cells, Auditory, Outer - metabolism
Hamsters
Health aspects
Histidine - metabolism
Molecular motors
Molecular weight
Monoclonal antibodies
Ovaries
Patch-Clamp Techniques
Personal computers
Physical Sciences
Protein structure
Proteins
Research and analysis methods
Software
Structure-function relationships
Sulfate Transporters - genetics
Sulfate Transporters - metabolism
Transfection
Variance analysis
Western blotting
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0314517