Affinity purification of the outer hair cell motor protein prestin using His-tag

Abstract

Objective

The high sensitivity and broad frequency selectivity of mammalian hearing are associated with the somatic motility of outer hair cells (OHCs) in the cochlea. This motility is considered to be induced by conformational changes of the motor protein prestin expressing in the lateral plasma membrane of OHCs. Since its identification in 2000, prestin has been actively investigated and its structure and function have gradually been elucidated. These successes are partly due to the development of efficient expression and purification system of the membrane proteins including prestin. To obtain further understandings of prestin, the development of various types of such systems will be essential. However, recent study protocols on membrane proteins have often employed HEK293 cells and have become complexed with expression genes carrying several proteins and peptides for stabilization and purification of the expressed proteins. In the present study, a simple expression and purification system using Chinese hamster ovary (CHO) cells and Hi-tag was developed.

Methods

Full length gerbil prestin was transfected into modified mammalian expression vectors with C-terminal 6 × His-tag. After drug selection with G418 for 4 weeks, single colonies were isolated by limiting dilution method. Cell lines highly expressing prestin were selected (named 3D5, 4D7 and 3C8). These cells were gently disrupted using a Dounce tissue grinder. Membrane fractions were extracted by ultracentrifugation and affinity chromatography was performed. The efficiency of the purification process was evaluated by quantitative Western blotting using a standard protein.

Results

Among the cell lines constructed, Western blotting analysis showed bands at around 100 kDa and the highest intensity was confirmed from the 3C8 cell line, indicating that this cell line has the highest expression of prestin molecules. The membrane fraction was therefore extracted from this cell line and subjected to the following purification procedure. It was found that 78.7 μg of prestin was purified from 2.0 × 10⁹ CHO cells.

Conclusion

In the present study, 78.7 μg of prestin was purified from 2.0 × 10⁹ CHO cells, which stably expressing 6 × His-tagged prestin, by extracting cell membrane fractions and standard affinity chromatography for His-tag.