Crystal structure analysis of MFS transporter proteins

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We have tried to construct expression systems for 29 proteins of multidrug export system MFS, SMR and MATE. Finally, expression and purification systems were established for three of them, QacA and NorA from Staphylococcus aureus, and CGL2611 from Corynebacterium glutamicum. We have also tried to use a Mistic fusion expression system, and have confirmed expression of EbrA, EbrB, TetB (13 transmembrane helices) and LmrB (14 transmembrane helices) .A detailed condition of preparing crystallization sample was examined for QacA, NorA and CGL2611. Effect of adding putative substrate was confirmed using Transmission Electron Microscope, CD measurement and Differential Scanning Calorimetry. For QacA, it was suggested that putative substrate Rhodamine 6G avoid its aggregation and three-dimensional structure was also stabilized. In the case of CGL2611, however, detergent DDM was troubling in a concentration process, and no putative substrate effective for its preparation was found.Phase diagram for solutions consist of 5 detergents and two precipitants, PEG400 and PEG3350, was observed, and initial crystallization conditions were established. With such informations, screening for initial crystallization conditions was done for QacA and NorA using a vapor diffusion method and a lipidic-cubic phase method at several temperatures. Some crystalline objects were observed, but no diffracting crystal was obtained