Molecular analysis of staphylococcal multidrug transport protein QacA

Abstract

The multidrug exporter QacA from Staphylococcus aureus confers resistance to a wide range of structurally-dissimilar monovalent and bivalent lipophilic, cationic compounds, including intercalating dyes, quaternary ammonium compounds (Qacs), diamidines, and biguanidines, many of which are used as antiseptics and disinfectants in current applications. To overcome such a problem, detailed understanding of the substrate-recognition and transport mechanisms of QacA is crucial. High-resolution structural studies of QacA can provide critical insights to these mechanisms. The preliminary requirement of such studies is a regular supply of purified QacA protein in milligram quantities. In this study, an over-expression and purification system of QacA based on the E. coli expression vector pTTQ18His6 was established. Conditions for the over-expression and purification of the QacA protein were optimized, resulting in a yield of approximately 600 pg of purified protein from 1 litre of bacterial culture. Circular dichroism (CD) spectroscopic analysis suggested that the purified QacA protein was structurally integral as demonstrated by its predominantly oc-helical structure, and substrate-binding assays were also performed.