Functional Expression of Prestin, the Outer Hair Cell Motor Protein, Using the Baculovirus/Insect Cell System

Abstract

A source of the electromotility of the outer hair cells (OHCs), which enables the high sensitivity of mammalian hearing, is believed to be voltage-dependent conformational change of the motor protein prestin embedded in the lateral wall of the OHCs. As a result of extensive studies on this unique motor protein using prestin-expressing cells, knowledge about its structure and function has been gradually accumulated. To obtain further knowledge about prestin, research using purified prestin molecules is necessary, and thus a method for efficiently obtaining prestin molecules is required. In the present study, construction of an expression system for prestin using the baculovirus/Sf9 insect cell system was attempted. The expression and localization in the plasma membrane of prestin in infected Sf9 cells were confirmed by Western blotting and immunofluorescence analysis, and prestin's functional activity was determined by patch-clamp measurements. Furthermore, to obtain prestin from the cells efficiently, culture conditions of the cells were examined, and it was clarified that cells should be harvested around 72 hours after infection. Quantitative Western blotting revealed that the maximal amount of prestin per liter of culture medium was around 800 µg. The results of this study indicate that the expression system which was constructed is potentially useful for purification of prestin due to the rapidity of its construction and its high yield.

The V499G/Y501H Mutation Impairs Fast Motor Kinetics of Prestin and Has Significance for Defining Functional Independence of Individual Prestin Subunits

“…If this were the case, 499-prestin subunits might not be readily interchangeable with WT-prestin. To test this idea, we examined heteromer formation between WT-and 499-prestin in a baculovirus/sf9 heterologous expression system, which attains a high efficacy of coexpression in sf9 cells that are known to produce functional prestin (29). Epitope-tagged prestin constructs were used because antibodies that distinguish 499-from WT-prestin are not available.…”